Pharmaceutical composition for treatment and/or prevention of cancer

ABSTRACT

This invention provides an antibody targeting a cancer antigenic protein specifically expressed on the surface of cancer cells and use thereof in a therapeutic and/or preventive agent for cancers. Specifically, this invention provides an antibody or a fragment thereof which has immunological reactivity with a partial CAPRIN-1 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 or an amino acid sequence having 80% or higher sequence identity to the amino acid sequence, and a pharmaceutical composition for treatment and/or prevention of cancers, comprising the antibody or fragment thereof as an active ingredient.

TECHNICAL FIELD

The present invention relates to novel use of an antibody againstCAPRIN-1 or a fragment thereof in a drug such as a therapeutic and/orpreventive agent for cancer.

BACKGROUND ART

Cancer is the leading cause of death. This disease is currently treatedprincipally by surgical therapy in combination with radiation therapyand/or chemotherapy. In spite of recent development of novel surgicaltechniques or discovery of novel anticancer agents, the existingtreatment of cancer has an insufficiently improved outcome, except forsome cancer types. With recent advances of molecular biology or cancerimmunology, antibodies that specifically react with cancer, cancerantigens that are recognized by cytotoxic T cells, genes encoding suchcancer antigens, and the like have been identified, raising expectationson specific cancer therapy targeting the cancer antigens (Non PatentLiterature 1).

For reducing the adverse reaction of cancer therapy, it is desired thatpeptides, polypeptides, or proteins recognized as antigens of the cancershould rarely exist in normal cells and specifically exist in cancercells. In 1991, Boon et al. (Ludwig Institute for Cancer Research,Belgium) isolated a human melanoma antigen MAGE1 recognized byCD8-positive T cells by a cDNA expression cloning method usingautologous cancer cell lines and cancer-reactive T cells (Non PatentLiterature 2). Then, a SEREX (serological identification of antigens byrecombinant expression cloning) method has been reported, which adopts agene expression cloning approach to identify tumor antigens recognizedby antibodies produced in response to autologous cancer in vivo in acancer patient (Non Patent Literature 3 and Patent Literature 1).According to this method, some cancer antigens that are rarely expressedin normal cells and are specifically expressed in cancer have beenisolated (Non Patent Literatures 4 to 9). In addition, cell therapyusing immunocytes that specifically react with cancer antigens orcancer-specific immunotherapy using vaccines or the like comprisingcancer antigens is under clinical trial targeting some of the isolatedcancer antigens.

In recent years, various antibody drugs for cancer treatment targetingantigenic proteins on cancer cells have emerged in the world. Thesedrugs have received attention because of their certain efficacy ascancer-specific therapeutic agents. A large majority of antigenicproteins targeted by the drugs, however, are also expressed in normalcells. As a result of administering the antibodies, cancer cells as wellas normal cells expressing the antigens are damaged, disadvantageouslyresulting in adverse reaction. Thus, if cancer antigens specificallyexpressed on the surface of cancer cells can be identified andantibodies targeting the antigens can be used as drugs, these antibodydrugs can be expected to achieve treatment with less adverse reaction.

Cytoplasmic- and proliferation-associated protein 1 (CAPRIN-1) has beenknown as an intracellular protein that is expressed upon activation orcell division of resting normal cells and forms cytoplasmic stressgranules with intracellular RNAs to participate in the regulation oftransport and translation of mRNAs. This protein has been found to bespecifically expressed on the surface of cancer cells and is under studyas a target of antibody drugs for cancer treatment (Patent Literature2).

PRIOR ART LITERATURE Patent Literature

-   Patent Literature 1: U.S. Pat. No. 5,698,396-   Patent Literature 2: WO2010/016526

Non Patent Literature

-   Non Patent Literature 1: Tsuyoshi Akiyoshi, “Japanese Journal of    Cancer and Chemotherapy”, 1997, Vol. 24, p. 55-519 (Japanese Journal    of Cancer and Chemotherapy Publishers Inc., Japan)-   Non Patent Literature 2: Bruggen P. et al., Science, 254: 1643-1647    (1991)-   Non Patent Literature 3: Proc. Natl. Acad. Sci. USA, 92: 11810-11813    (1995)-   Non Patent Literature 4: Int. J. Cancer, 72: 965-971 (1997)-   Non Patent Literature 5: Cancer Res., 58: 1034-1041 (1998)-   Non Patent Literature 6: Int. J. Cancer, 29: 652-658 (1998)-   Non Patent Literature 7: Int. J. Oncol., 14: 703-708 (1999)-   Non Patent Literature 8: Cancer Res., 56: 4766-4772 (1996)-   Non Patent Literature 9: Hum. Mol. Genet 6: 33-39, 1997

SUMMARY OF INVENTION Problem to be Solved by Invention

An object of the present invention is to produce an antibody thattargets CAPRIN-1 specifically expressed on the surface of cancer cellsand is superior in antitumor activity to conventional antibodies, and toprovide use thereof as a therapeutic and/or preventive agent for cancer.

Means for Solving Problem

Features of the present invention are as follows:

The present invention provides an antibody or a fragment thereof whichhas immunological reactivity with a partial CAPRIN-1 polypeptide havingthe amino acid sequence represented by SEQ ID NO: 5 or an amino acidsequence having 80% or higher sequence identity to the amino acidsequence, and a pharmaceutical composition for treatment and/orprevention of cancer, comprising the antibody or fragment thereof as anactive ingredient.

In the above embodiment, the cancer is breast cancer, kidney cancer,pancreatic cancer, colorectal cancer, lung cancer, brain tumor, gastriccancer, uterine cervix cancer, ovary cancer, prostate cancer, bladdercancer, esophageal cancer, leukemia, lymphoma, fibrosarcoma,mastocytoma, or melanoma.

In one embodiment, the antibody is a monoclonal antibody or a polyclonalantibody.

In another embodiment, the antibody is a human antibody, a humanizedantibody, a chimeric antibody, a single-chain antibody, or amultispecific antibody (e.g., a bispecific antibody).

The present specification encompasses the contents described in thespecification and/or drawings of Japanese Patent Application No.2012-035484 from which the present application claims the priority.

Effect of Invention

The antibody against CAPRIN-1 according to the present invention damagescancer cells. Thus, the antibody against CAPRIN-1 is useful in thetreatment or prevention of cancers.

MODE FOR CARRYING OUT INVENTION

The antibody according to the present invention is an antibody thatrecognizes and binds to a predetermined partial polypeptide of CAPRIN-1and has antitumor activity. More specifically, the antibody according tothe present invention is an antibody that recognizes (i.e., hasimmunological reactivity with) a partial polypeptide of a CAPRIN-1protein (partial CAPRIN-1 polypeptide) consisting of the amino acidsequence represented by SEQ ID NO: 5 or an amino acid sequence having80% or higher, preferably 85% or higher, more preferably 90% or higher,further preferably 95% or higher sequence identity to the amino acidsequence of SEQ ID NO: 5. In the present invention, it is revealed thatthis antibody exhibits antitumor activity. The present invention relatesto all antibodies that bind to fragments of CAPRIN-1 proteins asdescribed above and exhibit antitumor activity.

The antibody against CAPRIN-1 according to the present invention may beany type of antibody, with the proviso that it can exert antitumoractivity, and includes, for example, recombinant antibodies (e.g.,synthetic antibodies, multispecific antibodies (e.g., bispecificantibodies), humanized antibodies, chimeric antibodies, and single-chainantibodies (scFv)), human antibodies, and antibody fragments thereof(e.g., Fab, F(ab′)₂, and Fv). These antibodies and fragments thereof canbe prepared by methods generally known to those skilled in the art.Desirably, the antibody according to the present invention hasimmunological reactivity with a CAPRIN-1 protein or a partialpolypeptide thereof, or binds to (preferably, specifically binds to) theCAPRIN-1 protein through antigen-antibody reaction. As used herein, theterm “specifically binding to the CAPRIN-1 protein” means that theantibody specifically binds to the CAPRIN-1 protein withoutsubstantially binding to other proteins. The antibody according to thepresent invention is preferably a monoclonal antibody and however, maybe a polyclonal antibody as long as homogeneous antibodies can be stablyproduced. In the case that the subject is a human, human antibodies orhumanized antibodies are desirable for avoiding or suppressingrejection.

The antibody against a CAPRIN-1 polypeptide according to the presentinvention can be evaluated for its antitumor activity, as describedlater, by examining in vivo the inhibition of tumor growth in acancer-bearing animal or by examining in vitro whether or not theantibody exhibits immunocyte- or complement-mediated cytotoxic activityagainst tumor cells expressing the polypeptide.

The subject in need of the treatment and/or prevention of canceraccording to the present invention is a mammal such as a human, a petanimal, livestock, or a sport animal, preferably a human.

Hereinafter, the present invention will be described in more detail.

<Preparation of Antigen for Antibody Preparation>

Proteins or fragments thereof used as sensitizing antigens for obtainingthe antibody against CAPRIN-1 according to the present invention are notlimited by animal species serving as their origins, including humans,dogs, cats, cattle, horses, mice, rats, and chickens. The proteins orthe fragments thereof, however, are preferably selected in view ofcompatibility with parent cells for use in cell fusion. In general,mammal-derived proteins are preferred. Particularly, human-derivedproteins are preferred. For example, when CAPRIN-1 is human CAPRIN-1,human CAPRIN-1 protein, partial peptides thereof, or cells expressinghuman CAPRIN-1 can be used.

The nucleotide sequences and amino acid sequences of human CAPRIN-1 andhomologs thereof can be obtained, for example, by making an access toGenBank (NCBI, USA) and using an algorithm such as BLAST or FASTA(Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993;and Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997).

In the present invention, with reference to the nucleotide sequence (SEQID NO: 1 or 3) or the amino acid sequence (SEQ ID NO: 2 or 4) of humanCAPRIN-1, the target CAPRIN-1 is a nucleic acid or a protein consistingof a sequence having 70% to 100%, preferably 80% to 100%, morepreferably 90% to 100%, further preferably 95% to 100%, for example, 97%to 100%, 98% to 100%, 99% to 100%, or 99.5% to 100% sequence identity tothe nucleotide sequence or amino acid sequence of the ORF or matureportion of the reference (Note: when compared with each other, the aminoacid sequences of SEQ ID NO: 2 and SEQ ID NO: 4 differ in amino acidresidues at position 690 and following position 690). As used herein,the term “% sequence identity” means a percentage (%) of the number ofidentical amino acids (or nucleotides) to the total number (includingthe number of gaps) of amino acids (or nucleotides) when two sequencesare aligned such that the maximum degree of similarity or identity isachieved with or without gaps introduced.

A fragment that comprises an epitope (or an antigenic determinant),which is a minimal unit recognized by the antibody, and has a lengthranging from the amino acid length of the epitope to less than thefull-length of the CAPRIN-1 protein can be used as a CAPRIN-1 proteinfragment. The epitope refers to a polypeptide fragment havingantigenicity or immunogenicity in mammals, preferably humans. Itsminimal unit consists of approximately 7 to 12 amino acids, for example,8 to 11 amino acids. The CAPRIN-1 protein fragment for use in thepreparation of the antibody according to the present invention ispreferably a fragment that is recognized by the antibody of the presentinvention and comprises the amino acid sequence represented by SEQ IDNO: 5 (which corresponds to a sequence from positions 141 to 156 in theamino acid sequence of SEQ ID NO: 2 or 4) or an amino acid sequencehaving 80% or higher, preferably 85% or higher, more preferably 90% orhigher, further preferably 95% or higher sequence identity to the aminoacid sequence of SEQ ID NO: 5, or comprises at least an epitopeconsisting of approximately 7 to 12 consecutive amino acids, forexample, 8 to 11 consecutive amino acids in any of these amino acidsequences.

The above human CAPRIN-1 proteins and polypeptide fragments comprisingpartial peptides thereof can be synthesized according to chemicalsynthesis methods, for example, Fmoc (fluorenylmethyloxycarbonyl) andtBoc (t-butyloxycarbonyl) methods (Seikagaku Jikken Koza (BiochemicalExperimentation Course) 1, the Japanese Biochemical Society ed., ProteinChemistry IV, Chemical Modification and Peptide Synthesis, Tokyo KagakuDojin Co., Ltd. (Japan), 1981). Also, these polypeptides can besynthesized by routine methods using various commercially availablepeptide synthesizers.

Alternatively, polynucleotides encoding the polypeptides may be preparedusing genetic engineering techniques known in the art (Sambrook et al.,Molecular Cloning, the 2nd edition, Current Protocols in MolecularBiology (1989), Cold Spring Harbor Laboratory Press; Ausubel et al.,Short Protocols in Molecular Biology, the 3rd edition, A compendium ofMethods from Current Protocols in Molecular Biology (1995), John Wiley &Sons; etc.) and incorporated into expression vectors, which are thenintroduced into host cells so that the host cells produce thepolypeptides. In this way, the human CAPRIN-1 proteins of interest orthe polypeptide fragments thereof can be obtained.

The polynucleotides encoding the polypeptides can be readily prepared bygenetic engineering techniques known in the art or routine methods usingcommercially available nucleic acid synthesizers. For example, a DNAcomprising the nucleotide sequence of a human CAPRIN-1 gene can beprepared by PCR using a human chromosomal DNA or cDNA library as atemplate and a pair of primers designed so as to be capable ofamplifying the nucleotide sequence. Reaction conditions for this PCR canbe appropriately determined Examples of the conditions can include, butare not limited to, 30 cycles each involving reaction steps of 94° C.for 30 seconds (denaturation), 55° C. for 30 seconds to 1 minute(annealing), and 72° C. for 2 minutes (elongation) using thermostableDNA polymerase (e.g., Taq polymerase or Pfu polymerase) and aMg²⁺-containing PCR buffer, followed by reaction at 72° C. for 7minutes. The PCR approach, conditions, etc. are described in, forexample, Ausubel et al., Short Protocols in Molecular Biology, the 3rdedition, A Compendium of Methods from Current Protocols in MolecularBiology (1995), John Wiley & Sons (particularly, Chapter 15).

Also, appropriate probes or primers can be prepared on the basis ofinformation about the nucleotide sequences of CAPRIN-1 genes and theamino acid sequences of CAPRIN-1 proteins, and can be used in thescreening of, for example, a human cDNA library, to isolate the desiredDNA. Preferably, such a cDNA library is produced from cells, organs, ortissues expressing CAPRIN-1 proteins. Examples of such cells or tissuesinclude cells or tissues derived from the testis or from cancers ortumors such as leukemia, breast cancer, lymphoma, brain tumor, lungcancer, pancreatic cancer, and colorectal cancer. These techniques,including the preparation of probes or primers, the construction of acDNA library, the screening of the cDNA library, and the cloning of thegene of interest, are known to those skilled in the art and can beperformed according to methods described in, for example, Sambrook etal., Molecular Cloning, the 2nd edition, Current Protocols in MolecularBiology (1989), and Ausubel et al. (ibid.). DNAs encoding the humanCAPRIN-1 proteins and the partial peptides thereof can be obtained fromthe DNA thus obtained.

The host cells to receive the expression vectors may be any cell capableof expressing the above polypeptides. Examples of prokaryotic cellsinclude, but are not limited to, E. coli. Examples of eukaryotic cellsinclude, but are not limited to: mammalian cells such as monkey kidneycells COS1 and Chinese hamster ovary cells CHO; a human embryonic kidneycell line HEK293; a mouse embryonic skin cell line NIH3T3; yeast cellssuch as budding yeast and fission yeast cells; silkworm cells; andXenopus egg cells.

In the case of using prokaryotic cells as the host cells, the expressionvectors used have an origin that permits replication in the prokaryoticcells, a promoter, a ribosomal binding site, a multicloning site, aterminator, a drug resistance gene, an auxotrophic complementary gene,etc. Examples of expression vectors for E. coli can include pUC series,pBluescript II, pET expression systems, and pGEX expression systems. TheDNAs encoding the above polypeptides can be incorporated into suchexpression vectors, with which prokaryotic host cells are thentransformed, followed by culture of the obtained transformants so thatthe polypeptides encoded by the DNAs are expressed in the prokaryotichost cells. In this respect, the polypeptides may be expressed as fusionproteins with other proteins.

In the case of using eukaryotic cells as the host cells, expressionvectors for eukaryotic cells having a promoter, a splicing region, apoly(A) addition site, etc. are used as the expression vectors. Examplesof such expression vectors can include pKA1, pCDM8, pSVK3, pMSG, pSVL,pBK-CMV, pBK-RSV, EBV, pRS, pcDNA3, and pYES2 vectors. In the same wayas above, the DNAs encoding the above polypeptides can be incorporatedinto such expression vectors, with which eukaryotic host cells are thentransformed, followed by culture of the obtained transformants so thatthe polypeptides encoded by the DNAs are expressed in the eukaryotichost cells. In the case of using expression vectors such as pIND/V5-His,pFLAG-CMV-2, pEGFP-N1, or pEGFP-C1, the polypeptides may be expressed asvarious fusion proteins tagged with His tag (e.g., (His)₆ to (His)₁₀),FLAG tag, myc tag, HA tag, GFP, or the like.

The expression vectors can be introduced into the host cells using wellknown methods such as electroporation, a calcium phosphate method, aliposome method, a DEAE dextran method, microinjection, viral infection,lipofection, and binding with cell-penetrating peptides.

The polypeptide of interest can be isolated and purified from the hostcells by a combination of separation techniques known in the art.Examples thereof include, but are not limited to, treatment with adenaturant (e.g., urea) or surfactant, ultrasonication, enzymaticdigestion, salting-out, solvent fractionation and precipitation,dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE,isoelectric focusing, ion-exchange chromatography, hydrophobicchromatography, affinity chromatography, and reverse-phasechromatography.

The antigens thus prepared can be used as sensitizing antigens asdescribed later for producing the antibody according to the presentinvention.

<Structure of Antibody>

Antibodies (immunoglobulins) are usually heteromultimeric glycoproteinseach comprising at least two heavy chains and two light chains. Theimmunoglobulins, except for IgM, are heterotetrameric glycoproteins ofapproximately 150 kDa each composed of two identical light (L) chainsand two identical heavy (H) chains. Typically, each light chain isconnected to a heavy chain via a single disulfide covalent bond, whilethe number of disulfide bonds between heavy chains varies amongdifferent immunoglobulin isotypes. Each of the heavy and light chainsalso has an intrachain disulfide bond. Each heavy chain has a variabledomain (VH region) at one end, followed by a series of constant regions.Each light chain has a variable domain (VL region) at one end and has asingle constant region at the other end. The light chain constant regionis aligned with the first heavy chain constant region, while the lightchain variable domain is aligned with the heavy chain variable domain.Particular regions called complementarity determining regions (CDRs) inthe antibody variable domains exhibit specific variability and impartbinding specificity to the antibody. Portions relatively conserved inthe variable regions are called framework regions (FRs). The completeheavy and light chain variable domains each comprise four FRs connectedvia three CDRs. These three CDRs are called CDRH1, CDRH2, and CDRH3 inthis order from the N-terminus of the heavy chain. Likewise, the CDRsare called CDRL1, CDRL2, and CDRL3 in the light chain. CDRH3 is the mostimportant in view of the binding specificity of the antibody for itsantigen. In addition, CDRs in each chain are kept close to each othervia the FR regions and contribute to the formation of an antigen-bindingsite in the antibody, together with CDRs in the other chain. Theconstant regions do not directly contribute to antibody-antigen binding,but exhibit various effector functions, for example, involvement inantibody-dependent cellular cytotoxicity (ADCC), phagocytosis mediatedby binding to an Fcγ receptor, half-life/clearance rate mediated by aneonatal Fc receptor (FcRn), and complement-dependent cytotoxicity (CDC)mediated by a C1q component in the complement cascade.

<Preparation of Antibody>

The anti-CAPRIN-1 antibody according to the present invention means anantibody having immunological reactivity with a full-length CAPRIN-1protein or a fragment thereof. Particularly, the anti-CAPRIN-1 antibodyof the present invention is an antibody immunologically binding to apartial polypeptide of a CAPRIN-1 protein (partial CAPRIN-1 polypeptide)that is a peptide consisting of the epitope-containing amino acidsequence represented by SEQ ID NO: 5 or a polypeptide consisting of anamino acid sequence having 80% or higher, preferably 85% or higher, morepreferably 90% or higher, further preferably 95% or higher sequenceidentity to the amino acid sequence. Preferably, the antibody of thepresent invention recognizes an epitope consisting of approximately 7 to12 consecutive amino acids, for example, 8 to 11 consecutive aminoacids, in the amino acid sequence represented by SEQ ID NO: 5 or theamino acid sequence having 80% or higher, preferably 85% or higher, morepreferably 90% or higher, further preferably 95% or higher sequenceidentity to the amino acid sequence of SEQ ID NO: 5. This anti-CAPRIN-1antibody of the present invention is capable of specifically binding tothe full-length CAPRIN-1 protein. The antibody of the present inventioncan be obtained by selecting an antibody immunologically binding to thepolypeptide consisting of the amino acid sequence represented by SEQ IDNO: 5 or the polypeptide consisting of the amino acid sequence having80% or higher, preferably 85% or higher, more preferably 90% or higher,further preferably 95% or higher sequence identity to the amino acidsequence of SEQ ID NO: 5, according to a routine method from amongantibodies obtained with CAPRIN-1 proteins or fragments thereof asantigens.

As used herein, the term “immunological reactivity” means the propertyof the antibody binding to a CAPRIN-1 antigen (a full-length CAPRIN-1protein or a partial polypeptide thereof) in vivo. Via such binding toCAPRIN-1, the antibody of the present invention exerts the function ofdamaging (e.g., killing, suppressing, or regressing) tumor cells. Theantibody of the present invention can damage a tumor, for example,breast cancer, kidney cancer, pancreatic cancer, colorectal cancer(e.g., colon cancer), lung cancer, brain tumor, gastric cancer, uterinecervix cancer, ovary cancer, prostate cancer, bladder cancer, esophagealcancer, leukemia, lymphoma, fibrosarcoma, mastocytoma, or melanoma,through binding to the CAPRIN-1 protein.

The antibody of the present invention may be any type of antibody.Examples of the type of the antibody of the present invention includemonoclonal antibodies, polyclonal antibodies, synthetic antibodies,multispecific antibodies, human antibodies, humanized antibodies,chimeric antibodies, single-chain antibodies, and antibody fragments(e.g., Fab, F(ab′)₂, and Fv). Also, the antibody is any class ofimmunoglobulin molecule, for example, IgG, IgE, IgM, IgA, IgD, or IgY,or any subclass, for example, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

The antibody may be further modified by a modification such asacetylation, formylation, amidation, phosphorylation, PEGylation, or thelike, in addition to glycosylation.

Hereinafter, preparation examples of various antibodies will bedescribed.

When the antibody of the present invention is a monoclonal antibody, forexample, a breast cancer cell line SK-BR-3 expressing CAPRIN-1 isadministered to each mouse for immunization. The spleen is removed fromthis mouse. After separation of spleen cells, the cells are fused withmouse myeloma cells. Clones producing antibodies having a cancer cellgrowth inhibitory effect are selected from among the obtained fusioncells (hybridomas). Alternatively, clones producing antibodies bindingto a polypeptide consisting of the amino acid sequence represented bySEQ ID NO: 5 or to a polypeptide consisting of an amino acid sequencehaving 80% or higher sequence identity to the amino acid sequence of SEQID NO: 5 may be selected. The hybridomas producing monoclonal antibodieshaving a cancer cell growth inhibitory effect or the hybridomasproducing monoclonal antibodies against the polypeptide of SEQ ID NO: 5or the like are isolated and cultured. The antibody of the presentinvention can be prepared by purification from the culture supernatantaccording to a general affinity purification method.

The monoclonal antibody-producing hybridomas may be prepared, forexample, as follows: first, animals are immunized with sensitizingantigens according to a method known in the art. This immunizationmethod generally involves intraperitoneally or subcutaneously injectingthe sensitizing antigens to mammals. Specifically, the sensitizingantigens are diluted with or suspended in PBS (phosphate-bufferedsaline), physiological saline, or the like into an appropriate amountand then mixed, if desired, with an appropriate amount of a conventionaladjuvant, for example, a complete Freund's adjuvant. Afteremulsification, the resulting emulsion is administered to each mammalseveral times every 4 to 21 days. Alternatively, an appropriate carriermay be used for the immunization with sensitizing antigens.

After confirmation of a rise in the level of the desired antibody in theserum of the mammal thus immunized, immunocytes are collected from themammal and subjected to cell fusion. Preferred examples of theimmunocytes particularly include spleen cells.

Mammalian myeloma cells are used as partner parent cells to be fusedwith the immunocytes. Various cell lines known in the art, for example,P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979) 123,1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology(1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol.(1976) 6, 511-519), MPC-11 (Margulies. D. H. et al., Cell (1976) 8,405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO(deSt. Groth, S. F. et al., J. Immunol. Methods (1980) 35, 1-21), 5194(Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), and R210 (Galfre,G. et al., Nature (1979) 277, 131-133), are preferably used as themyeloma cells.

The cell fusion between the immunocytes and the myeloma cells can beperformed basically according to a method known in the art, for example,the method of Kohler and Milstein (Kohler, G. and Milstein, C. MethodsEnzymol. (1981) 73, 3-46).

More specifically, the cell fusion is carried out, for example, in thepresence of a cell fusion promoter in a conventional nutrient medium.For example, polyethylene glycol (PEG) or Sendai virus (hemagglutinatingvirus of Japan, HVJ) is used as the fusion promoter. If desired, anauxiliary such as dimethyl sulfoxide may be further added in order toenhance fusion efficiency.

The ratio between the immunocytes and the myeloma cells used can bearbitrarily set. For example, the amount of the immunocytes ispreferably set to 1 to 10 times the amount of the myeloma cells.Examples of the medium that can be used in the cell fusion includeRPMI1640 and MEM media suitable for the growth of the myeloma cell linesas well as conventional media for use in this type of cell culture. Inaddition, a serum supplement such as fetal calf serum (FCS) may be usedin combination with these media.

For the cell fusion, the immunocytes and the myeloma cells are wellmixed in their predetermined amounts in the medium. A PEG solution(average molecular weight: for example, approximately 1,000 to 6,000)preheated to approximately 37° C. is usually added to the mixture at aconcentration of 30 to 60% (w/v) and mixed therewith to form thehybridomas of interest. Subsequently, procedures of sequentially addingan appropriate medium and removing the supernatant by centrifugation arepreferably repeated to remove cell fusion agents or the like unfavorablefor the growth of the hybridomas.

The hybridomas thus obtained are cultured in a conventional selectivemedium, for example, a HAT medium (which contains hypoxanthine,aminopterin, and thymidine) for selection. This culture in the HATmedium is continued for a period (usually, several days to severalweeks) sufficient for the death of cells (non-fused cells) other thanthe hybridomas of interest. Subsequently, hybridomas producing theantibody of interest are screened for and cloned as single clones by aconventional limiting dilution method.

In addition to obtaining the hybridomas by immunization of non-humananimals with antigens, hybridomas producing human antibodies having thedesired activity (e.g., cell growth inhibitory activity) may be obtainedby sensitizing human lymphocytes, for example, EB virus-infected humanlymphocytes, with proteins, protein-expressing cells, or lysates thereofin vitro and fusing the sensitized lymphocytes with human-derivedmyeloma cells capable of dividing permanently, for example, U266(Registration No. TIB196).

The monoclonal antibody-producing hybridomas thus prepared can besubcultured in a conventional medium and can also be stored for a longperiod in liquid nitrogen.

Specifically, the desired antigens or cells expressing the desiredantigens are used as sensitizing antigens in immunization according to aconventional immunization method. The obtained immunocytes are fusedwith parent cells known in the art according to a conventional cellfusion method. Monoclonal antibody-producing cells (hybridomas) can bescreened for by a conventional screening method to prepare the antibodyof interest.

Another example of the antibody that may be used in the presentinvention is a polyclonal antibody. The polyclonal antibody can beobtained, for example, as follows:

Serum is obtained from small animals such as mice, humanantibody-producing mice, or rabbits immunized with natural CAPRIN-1proteins or recombinant CAPRIN-1 proteins expressed as fusion proteinswith GST or the like in microorganisms such as E. coli, or partialpeptides thereof. Alternatively, serum may be obtained from mammalsimmunized with CAPRIN-1 fragments serving as sensitizing antigens, i.e.,a polypeptide comprising the amino acid sequence represented by SEQ IDNO: 5 or an amino acid sequence having 80% or higher, preferably 85% orhigher, more preferably 90% or higher, further preferably 95% or highersequence identity to the amino acid sequence of SEQ ID NO: 5(preferably, a polypeptide consisting of any of these amino acidsequences), or a polypeptide comprising an epitope (preferably,consisting of the epitope) consisting of approximately 7 to 12consecutive amino acids, for example, 8 to 11 consecutive amino acids,in the amino acid sequence represented by SEQ ID NO: 5 or the amino acidsequence having 80% or higher, preferably 85% or higher, more preferably90% or higher, further preferably 95% or higher sequence identity to theamino acid sequence of SEQ ID NO: 5. The serum thus obtained can bepurified using, for example, ammonium sulfate precipitation, protein Aor protein G columns, DEAE ion-exchange chromatography, or affinitycolumns coupled with CAPRIN-1 proteins or synthetic peptides to prepareanti-CAPRIN-1 polyclonal antibodies. The polyclonal antibody of thepresent invention includes antibodies obtained from humanantibody-producing animals (e.g., mice) immunized with CAPRIN-1 protein.

In this context, for example, KM mice (Kirin Pharma Co., Ltd./Medarex)and Xeno mice (Amgen Inc.) are known as the human antibody-producingmice (e.g., International Publication Nos. WO02/43478 and WO02/092812).Complete human polyclonal antibodies can be obtained from the blood ofsuch mice immunized with CAPRIN-1 protein or fragments thereof.Alternatively, spleen cells may be isolated from the mice thus immunizedand fused with myeloma cells. In this way, human monoclonal antibodiescan be obtained.

The antigens can be prepared according to, for example, a method usinganimal cells (JP Patent Publication (Kohyo) No. 2007-530068 A (2007)) ora method using baculovirus (e.g., International Publication No.WO98/46777). Antigens having low immunogenicity can be bound toimmunogenic macromolecules such as albumin for immunization. Theantigens may be administered together with adjuvants for immunization.

Alternatively, the antibody of the present invention may be obtained asa genetically recombinant antibody that is produced using a generecombination technique which involves: cloning a gene of the antibodyfrom hybridomas; incorporating the antibody gene into appropriatevectors; and introducing the vectors into hosts (see, e.g., Carl, A. K.Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES,Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).Specifically, antibody variable region (V region) cDNAs are synthesizedfrom the mRNAs of hybridomas using reverse transcriptase. Afterobtainment of DNAs encoding the antibody V regions of interest, the DNAsare ligated with DNAs encoding the desired antibody constant regions (Cregions). The resulting ligation products are incorporated intoexpression vectors. Alternatively, the antibody V region-encoding DNAsmay be incorporated into expression vectors containing antibody C regionDNAs. These DNAs are incorporated into the expression vectors so as tobe expressed under the control of expression control regions, forexample, an enhancer and a promoter. Next, host cells can be transformedwith the resulting expression vectors and allowed to express antibodies.

The anti-CAPRIN-1 antibody of the present invention is preferably amonoclonal antibody. Alternatively, the anti-CAPRIN-1 antibody of thepresent invention may be a polyclonal antibody, a genetically engineeredantibody (chimeric antibody, humanized antibody, etc.), or the like.

The monoclonal antibody includes human monoclonal antibodies, non-humananimal monoclonal antibodies (e.g., mouse, rat, rabbit, and chickenmonoclonal antibodies), chimeric monoclonal antibodies, and the like.The monoclonal antibody may be prepared by the culture of hybridomasobtained by the fusion between spleen cells from non-human mammals(e.g., mice, human antibody-producing mice, chickens, or rabbits)immunized with CAPRIN-1 proteins or fragments thereof and myeloma cells.The chimeric antibody is an antibody prepared from a combination ofsequences derived from different animals and is, for example, anantibody composed of mouse antibody heavy and light chain variableregions and human antibody heavy and light chain constant regions. Thechimeric antibody can be prepared using a method known in the art whichinvolves, for example: ligating DNAs encoding mouse antibody V regionswith DNAs encoding human antibody C regions; incorporating the resultingligation products into expression vectors; and introducing the vectorsinto hosts so that antibodies are produced.

Monoclonal antibodies that have immunological reactivity with thepartial CAPRIN-1 polypeptide consisting of the amino acid sequencerepresented by SEQ ID NO: 5 and have an antitumor effect are prepared bya method described later in Examples.

The humanized antibody, also called reshaped human antibody, is anengineered antibody. The humanized antibody is constructed by graftingcomplementarity determining regions (CDRs) of an antibody of animmunized animal at the corresponding complementarity determiningregions (CDRs) of a human antibody. A general gene recombinationapproach therefor is also known.

Specifically, DNA sequences designed so as to link, for example, mouse,rabbit, or chicken antibody CDRs, and human antibody framework regions(FRs) are synthesized by PCR from several prepared oligonucleotideshaving terminal portions overlapping with each other. The obtained DNAsare ligated with DNAs encoding human antibody constant regions.Subsequently, the resulting ligation products are incorporated intoexpression vectors, which are then introduced into hosts for antibodyproduction to obtain the antibody of interest (see European PatentApplication Publication No. EP239400 and International Publication No.WO96/02576). The human antibody FRs connected via complementaritydetermining regions (CDRs) are selected such that the CDRs form afavorable antigen-binding site. If necessary, amino acids in theframework regions of antibody variable regions may be substituted suchthat the complementarity determining regions of the resulting reshapedhuman antibody form an appropriate antigen-binding site (Sato K. et al.,Cancer Research 1993, 53: 851-856). In addition, these FRs may bereplaced with framework regions derived from human antibodies ofdifferent class or subclass (see International Publication No.WO99/51743).

Amino acids in variable regions (e.g., FRs) or constant regions of thechimeric antibody or the humanized antibody thus prepared may besubstituted, for example, by other amino acids.

The amino acid substitution is the substitution of, for example, lessthan 15, less than 10, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less amino acids, preferably 1 to 5 aminoacids, more preferably 1 or 2 amino acids. The substituted antibodyshould be functionally equivalent to an unsubstituted antibody. Thesubstitution is desirably conservative amino acid substitution, which isthe substitution between amino acids similar in properties such ascharge, side chains, polarity, and aromaticity. The amino acids can beclassified in terms of similar properties into, for example: basic aminoacids (arginine, lysine, and histidine); acidic amino acids (asparticacid and glutamic acid); uncharged polar amino acids (glycine,asparagine, glutamine, serine, threonine, cysteine, and tyrosine);nonpolar amino acids (leucine, isoleucine, alanine, valine, proline,phenylalanine, tryptophan, and methionine); branched amino acids(leucine, valine, and isoleucine); and aromatic amino acids(phenylalanine, tyrosine, tryptophan, and histidine).

Examples of modified antibodies can include antibodies bound withvarious molecules such as polyethylene glycol (PEG). In the modifiedantibody of the present invention, the substance to be bound is notlimited. In order to obtain such a modified antibody, the obtainedantibody can be chemically modified. A method therefor has already beenestablished in the art.

As used herein, the term “functionally equivalent” means that anantibody of interest has biological or biochemical activity similar tothat of the antibody of the present invention, specifically, theantibody of interest has the function of damaging tumor and essentiallycauses no rejection when applied to humans, for example. Examples ofsuch activity can include cell growth inhibitory activity and bindingactivity.

A method for preparing a polypeptide functionally equivalent to acertain polypeptide, which involves introducing a mutation into apolypeptide, is well known to those skilled in the art. For example,those skilled in the art can appropriately introduce a mutation into theantibody of the present invention using site-directed mutagenesis(Hashimoto-Gotoh, T. et al., (1995) Gene 152, 271-275; Zoller, M J., andSmith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al.,(1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W. and Fritz, H J.,(1987) Methods Enzymol. 154, 350-367; Kunkel, T A., (1985) Proc. Natl.Acad. Sci. USA. 82, 488-492; and Kunkel (1988) Methods Enzymol. 85,2763-2766) or the like, thereby preparing an antibody functionallyequivalent to the antibody of the present invention.

The antibody that recognizes an epitope of a CAPRIN-1 protein or aCAPRIN-1 fragment polypeptide comprising the epitope can be obtained bya method generally known to those skilled in the art. For example, theantibody can be obtained by a method which involves determining theepitope of the CAPRIN-1 protein recognized by the obtained anti-CAPRIN-1antibody having a cancer cell growth inhibitory effect by a conventionalmethod (e.g., epitope mapping or an epitope identification methoddescribed later) and preparing an antibody using a polypeptide having anamino acid sequence contained in the epitope as an immunogen, or amethod which involves determining an epitope for an antibody prepared bya conventional method and selecting an antibody that recognizes the sameepitope as that for the anti-CAPRIN-1 antibody. As used herein, the term“epitope” refers to a polypeptide fragment having antigenicity orimmunogenicity in mammals, preferably humans. Its minimal unit consistsof approximately 7 to 12 amino acids, preferably 8 to 11 amino acids.

The antibody of the present invention is an antibody havingimmunological reactivity with CAPRIN-1, an antibody that specificallyrecognizes CAPRIN-1, or an antibody that specifically binds to CAPRIN-1and exhibits cytotoxic activity against cancer or a tumor growthinhibitory effect. The antibody is preferably an antibody having astructure that causes little or no rejection in recipient animals.Examples of such antibodies include human antibodies, humanizedantibodies, chimeric antibodies (e.g., human-mouse chimeric antibodies),single-chain antibodies, and bispecific antibodies when the recipientanimals are humans. These antibodies have heavy and light chain variableregions derived from a human antibody or have heavy and light chainvariable regions with complementarity determining regions (CDR1, CDR2,and CDR3) derived from a non-human animal antibody and framework regions(FR1, FR2, FR3, and FR4) derived from a human antibody. Alternatively,these antibodies are recombinant antibodies having heavy and light chainvariable regions derived from a non-human animal antibody and heavy andlight chain constant regions derived from a human antibody. The antibodyof the present invention is preferably the former two antibodies.

Such recombinant antibodies can be prepared as follows: DNAs encodingmonoclonal antibodies (e.g., human, mouse, rat, rabbit, and chickenmonoclonal antibodies) against human CAPRIN-1 are cloned fromantibody-producing cells such as hybridomas and used as templates inRT-PCR or the like to prepare DNAs encoding the light and heavy chainvariable regions of the antibodies. The respective sequences of thelight and heavy chain variable regions, the respective sequences ofCDR1, CDR2, and CDR3 in each region, or the respective sequences of FR1,FR2, FR3, and FR4 in each region can be determined on the basis of, forexample, the Kabat EU numbering system (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institute of Health, Bethesda, Md. (1991)).

Such a DNA encoding each variable region or a DNA encoding each CDR isprepared using a gene recombination technique (Sambrook et al.,Molecular Cloning A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989)) or a DNA synthesizer. In this context, the humanmonoclonal antibody-producing hybridomas can be prepared by immunizinghuman antibody-producing animals (e.g., mice) with human CAPRIN-1 andthen fusing spleen cells excised from the immunized animals with myelomacells. Aside from this, DNAs encoding human antibody-derived light orheavy chain variable and constant regions are prepared, if necessary,using a gene recombination technique or a DNA synthesizer.

For the humanized antibody, DNAs in which the CDR coding sequences inDNAs encoding human antibody-derived light or heavy chain variableregions are substituted by corresponding CDR coding sequences of anon-human animal (e.g., mouse, rat, rabbit, or chicken)-derived antibodycan be prepared and ligated with the DNAs encoding humanantibody-derived light or heavy chain constant regions to prepare a DNAencoding the humanized antibody.

For the chimeric antibody, DNAs encoding light or heavy chain variableregions of a non-human animal (e.g., mouse, rat, rabbit, orchicken)-derived antibody can be ligated with DNAs encoding humanantibody-derived light or heavy chain constant regions to prepare a DNAencoding the chimeric antibody.

The single-chain antibody refers to an antibody comprising heavy andlight chain variable regions linearly linked to each other via a linker.A DNA encoding the single-chain antibody can be prepared by ligating aDNA encoding the heavy chain variable region, a DNA encoding the linker,and a DNA encoding the light chain variable region. In this context, theheavy and light chain variable regions are both derived from a humanantibody or derived from a human antibody having CDRs alone substitutedby CDRs of a non-human animal (e.g., mouse, rat, rabbit, orchicken)-derived antibody. The linker consists of 12 to 19 amino acids.Examples thereof include (G₄S)₃ consisting of 15 amino acids (G. B. Kimet al., Protein Engineering Design and Selection 2007, 20 (9): 425-432).

The bispecific antibody (e.g., diabody) refers to an antibody capable ofspecifically binding to two different epitopes. A DNA encoding thebispecific antibody can be prepared by ligating, for example, a DNAencoding a heavy chain variable region A, a DNA encoding a light chainvariable region B, a DNA encoding a heavy chain variable region B, and aDNA encoding a light chain variable region A in this order (providedthat the DNA encoding a light chain variable region B and the DNAencoding a heavy chain variable region B are ligated via a DNA encodinga linker as described above). In this context, the heavy and light chainvariable regions are all derived from a human antibody or derived from ahuman antibody having CDRs alone substituted by CDRs of a non-humananimal (e.g., mouse, rat, rabbit, or chicken)-derived antibody.

The recombinant DNAs thus prepared can be incorporated into one or moreappropriate vectors, which are then introduced into host cells (e.g.,mammalian cells, yeast cells, and insect cells) so that the DNAs are(co)expressed to produce recombinant antibodies (P. J. Delves, ANTIBODYPRODUCTION ESSENTIAL TECHNIQUES, 1997 WILEY, P. Shepherd and C. Dean,Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; and J. W. Goding,Monoclonal Antibodies: principles and practice, 1993 ACADEMIC PRESS).

Examples of the antibody of the present invention prepared by any of themethods described above include the following antibody (a) obtained inExamples described later:

(a) an antibody comprising a heavy chain variable region comprisingcomplementarity determining regions of SEQ ID NOs: 8, 9, and 10 and alight chain variable region comprising complementarity determiningregions of SEQ ID NOs: 12, 13, and 14 (e.g., an antibody constituted bya heavy chain variable region of SEQ ID NO: 11 and a light chainvariable region of SEQ ID NO: 15).

In this context, the amino acid sequences represented by SEQ ID NOs: 8,9, and 10 correspond to CDR1, CDR2, and CDR3 of the heavy chain variableregion of a mouse-derived antibody, respectively. The amino acidsequences represented by SEQ ID NOs: 12, 13, and 14 correspond to CDR1,CDR2, and CDR3 of the light chain variable region of a mouse-derivedantibody, respectively.

Examples of the humanized antibody, the chimeric antibody, thesingle-chain antibody, or the bispecific antibody of the presentinvention include the following antibodies (i) to (iii):

(i) an antibody comprising a heavy chain variable region comprisingCDR1, CDR2, and CDR3 consisting of the amino acid sequences of SEQ IDNOs: 8, 9, and 10, respectively, and human antibody-derived frameworkregions, and a light chain variable region comprising CDR1, CDR2, andCDR3 consisting of the amino acid sequences of SEQ ID NOs: 12, 13, and14, respectively, and human antibody-derived framework regions;

(ii) an antibody comprising a heavy chain comprising a heavy chainvariable region comprising CDR1, CDR2, and CDR3 consisting of the aminoacid sequences of SEQ ID NOs: 8, 9, and 10, respectively, and humanantibody-derived framework regions, and a human antibody-derived heavychain constant region, and a light chain comprising a light chainvariable region comprising CDR1, CDR2, and CDR3 consisting of the aminoacid sequences of SEQ ID NOs: 12, 13, and 14, respectively, and humanantibody-derived framework regions, and a human antibody-derived a lightchain constant region; and

(iii) an antibody comprising a heavy chain comprising a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 11 anda human antibody-derived heavy chain constant region, and a light chaincomprising a light chain variable region comprising the amino acidsequence of SEQ ID NO: 15 and a human antibody-derived light chainconstant region.

The sequences of the constant and variable regions of human antibodyheavy and light chains are available from, for example, NCBI (USA;GenBank, UniGene, etc.). For example, the following sequences can bereferred to: Registration No. J00228 for a human IgG1 heavy chainconstant region; Registration No. J00230 for a human IgG2 heavy chainconstant region; Registration No. X03604 for a human IgG3 heavy chainconstant region; Registration No. K01316 for a human IgG4 heavy chainconstant region; Registration Nos. V00557, X64135, X64133, etc. for ahuman light chain κ constant region; and Registration Nos. X64132,X64134, etc. for a human light chain λ constant region.

Preferably, these antibodies have cytotoxic activity and can therebyexert an antitumor effect.

The above particular sequences of the heavy and light chain variableregions and CDRs in each antibody are provided merely for illustrativepurposes. It is obvious that the antibody of the present invention isnot limited by the particular sequences. Hybridomas capable of producinganti-human CAPRIN-1 human antibodies or non-human animal antibodies(e.g., mouse antibodies) different from those described above areprepared, and monoclonal antibodies produced by the hybridomas arerecovered and assessed as being (or being not) the antibodies ofinterest with immunological binding activity against human CAPRIN-1 andcytotoxic activity as indicators. The monoclonal antibody-producinghybridomas of interest are thereby identified. Then, DNAs encoding theheavy and light chain variable regions of the antibodies of interest areproduced from the hybridomas and sequenced, as described above. The DNAsare used for the preparation of the different antibodies.

The above antibodies may each have the substitution, deletion, oraddition of one or several amino acids, particularly in a frameworkregion sequence and/or a constant region sequence, as long as theantibody has such specificity that it can specifically recognizeCAPRIN-1. As used herein, the term “several” means preferably 2 to 5,more preferably 2 or 3.

The affinity constant Ka (K_(on)/k_(off)) of the antibody of the presentinvention for a CAPRIN-1 protein or a fragment thereof is preferably atleast 10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 5×10⁸ M⁻¹, at least 10⁹ M⁻¹,at least 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 5×10¹⁰ M⁻¹, at least10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹² M⁻¹, or at least 10¹³ M⁻¹.

The antibody of the present invention can be conjugated with anantitumor agent. The conjugation of the antibody with the antitumoragent can be performed via a spacer having a group (e.g., a succinimidylgroup, a formyl group, a 2-pyridyldithio group, a maleimidyl group, analkoxycarbonyl group, or a hydroxy group) reactive with an amino group,a carboxyl group, a hydroxy group, a thiol group, or the like.

Examples of the antitumor agent include the following antitumor agentspublicly known in literatures, etc.: paclitaxel, doxorubicin,daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa,busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa,uredopa, altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, trimethylolomelamine, bullatacin,bullatacinone, camptothecin, bryostatin, callystatin, cryptophycin 1,cryptophycin 8, dolastatin, duocarmycin, eleutherobin, pancratistatin,sarcodictyin, spongistatin, chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine,calicheamicin, dynemicin, clodronate, esperamicin, aclacinomycin,actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin,carminomycin, carzinophilin, chromomycin, dactinomycin, detorbicin,6-diazo-5-oxo-L-norleucine, Adriamycin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin,olivomycin, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin, denopterin, pteropterin, trimetrexate,fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine, androgens (e.g., calusterone,dromostanolone propionate, epitiostanol, mepitiostane, andtestolactone), aminoglutethimide, mitotane, trilostane, frolinic acid,aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil,amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine,diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid,lentinan, lonidamine, maytansine, ansamitocin, mitoguazone,mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet,pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide,procarbazine, razoxane, rhizoxin, schizophyllan, spirogermanium,tenuazonic acid, triaziquone, roridin A, anguidine, urethane, vindesine,dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,gacytosine, docetaxel, chlorambucil, gemcitabine, 6-thioguanine,mercaptopurine, cisplatin, oxaliplatin, carboplatin, vinblastine,etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine,novantrone, teniposide, edatrexate, daunomycin, aminopterin, Xeloda,ibandronate, irinotecan, topoisomerase inhibitors,difluoromethylornithine (DMFO), retinoic acid, capecitabine, andpharmaceutically acceptable salts and derivatives thereof

Alternatively, the antibody of the present invention can be administeredin combination with an antitumor agent to produce a higher therapeuticeffect. This approach is applicable to a patient with cancer expressingCAPRIN-1 either before or after surgical operation. This approach can beapplied, particularly after surgery, to CAPRIN-1-expressing cancer,which has been treated conventionally with an antitumor agent alone, toproduce higher prevention of cancer recurrence or prolongation ofsurvival time.

Examples of the antitumor agent used in the combined administration withthe antibody of the present invention also include the followingantitumor agents publicly known in literatures, etc.: paclitaxel,doxorubicin, daunorubicin, cyclophosphamide, methotrexate,5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa,carboquone, meturedopa, uredopa, altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide,trimethylolomelamine, bullatacin, bullatacinone, camptothecin,bryostatin, callystatin, cryptophycin 1, cryptophycin 8, dolastatin,duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin,chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine, calicheamicin, dynemicin, clodronate,esperamicin, aclacinomycin, actinomycin, authramycin, azaserine,bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin,chromomycin, dactinomycin, detorbicin, 6-diazo-5-oxo-L-norleucine,Adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycinC, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin,trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine,ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone,dromostanolone propionate, epitiostanol, mepitiostane, testolactone,aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone,aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine,bestrabucil, bisantrene, edatraxate, defofamine, demecolcine,diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid,lentinan, lonidamine, maytansine, ansamitocin, mitoguazone,mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet,pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide,procarbazine, razoxane, rhizoxin, schizophyllan, spirogermanium,tenuazonic acid, triaziquone, roridin A, anguidine, urethane, vindesine,dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,gacytosine, docetaxel, chlorambucil, gemcitabine, 6-thioguanine,mercaptopurine, cisplatin, oxaliplatin, carboplatin, vinblastine,etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine,novantrone, teniposide, edatrexate, daunomycin, aminopterin, Xeloda,ibandronate, irinotecan, topoisomerase inhibitors,difluoromethylornithine (DMFO), retinoic acid, capecitabine, andpharmaceutically acceptable salts (known in the art) and derivatives(known in the art) thereof. Of these antitumor agents, cyclophosphamide,paclitaxel, docetaxel, or vinorelbine is particularly preferably used.

Alternatively, the antibody of the present invention may be bound to aradioisotope publicly known in literatures, etc., such as, ²¹¹At, ¹³¹I,¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ¹⁷⁵Lu, ¹⁷⁶Lu, ⁸⁹Sr, ⁶⁴Cu, or¹¹¹In (Hideo Saji, YAKUGAKU ZASSHI 128 (3) 323-332, 8 (2008), Jpn). Aradioisotope effective for the treatment or diagnosis of tumor isdesirable. Such a radioisotope is also included in the antitumor agentaccording to the present invention.

<Identification of Epitope>

As shown in Examples below, the antibody of the present invention bindsto an epitope in the amino acid sequence represented by SEQ ID NO: 5.One example of a method for confirming an epitope for the antibody ofthe present invention includes a method which involves immobilizing anepitope in the polypeptide of SEQ ID NO: 5 onto a plate and evaluatingthe antibody for its reactivity with this epitope. Specifically, anepitope in the polypeptide of SEQ ID NO: 5 is immobilized throughreaction onto a plate attached with electron-withdrawing functionalgroups via spacer such as oligoethylene glycol. The antibody of thepresent invention can be reacted with the plate and evaluated for itsreactivity with the epitope through reaction with a labeled (e.g.,horseradish peroxidase (HRP)-labeled) secondary antibody binding to theantibody of the present invention, i.e., the epitope to which theantibody of the present invention binds can be confirmed. The epitope inthe polypeptide of SEQ ID NO: 5 used in the immobilization onto a plateis a sequence itself comprising at least the epitope in the sequence ofSEQ ID NO: 5 or a modified portion thereof (e.g., N-terminal orC-terminal residues modified with several arbitrary amino acids or aprotein such as KLH or a (poly)peptide modified with a MAP protein). Theantibody of the present invention needs only to bind to any of these(poly)peptides.

On the other hand, even the antibody of the present invention may beunreactive with the polypeptide of SEQ ID NO: 5, i.e., the epitope maynot be confirmed, in the above method. In this case, the antibody isreacted with an antigen under solution conditions that facilitate tobind the antigen and the antibody. After obtainment of anantigen-antibody complex by an immunoprecipitation method, a partialpolypeptide bound with the antibody can be separated and examined forits amino acid sequence to confirm the epitope for the antibody of thepresent invention. In this context, the antigen is, for example, thepolypeptide of SEQ ID NO: 5 itself or a modified portion thereof.Alternatively, even a CAPRIN-1 protein may be used as long as theepitope reactive with the antibody of the present invention can beconfirmed by the above method.

<Antitumor Effect>

The antitumor effect of the anti-CAPRIN-1 antibody used in the presentinvention on CAPRIN-1-expressing cancer cells seems to be brought aboutby the following mechanism: effector cell-mediated antibody-dependentcellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)against the CAPRIN-1-expressing cells. However, this mechanism is notintended to limit the scope of the present invention.

The antitumor effect based on the mechanism is known to correlate withthe number of target molecules expressed on the surface of cancer cellsto which the antibody binds (Niwa R., Clinical Cancer Research 2005 Mar.15; 11 (6): 2327-2336). The number of target molecules expressed on thesurface of cancer cells can be examined using an existing assay kitcapable of measuring the number of cell surface molecules. Specifically,the number of target molecules to which the antibody binds can bedetermined by: reacting primary antibodies such as antibodies againstthe target molecules with cancer cells; reacting therewith fluorescentlylabeled antibodies against the primary antibodies together with beadsfor a calibration curve with the known number of molecules; andmeasuring the mean fluorescence intensity of the samples to obtain acalibration curve.

Thus, the anti-CAPRIN-1 antibody used in the present invention can beevaluated for its activity, as specifically shown in Examples below, byassaying the ADCC or CDC activity against CAPRIN-1-expressing cancercells in vitro or by examining the number of CAPRIN-1 moleculesexpressed on the surface of cancer cells using the anti-CAPRIN-1antibody according to the present invention as a primary antibody.

The anti-CAPRIN-1 antibody used in the present invention binds to aCAPRIN-1 protein on cancer cells and exhibits an antitumor effectthrough the activity. Thus, the anti-CAPRIN-1 antibody of the presentinvention is useful in the treatment or prevention of cancer. Thus, thepresent invention provides a pharmaceutical composition for treatmentand/or prevention of a cancer, comprising the anti-CAPRIN-1 antibody asan active ingredient. The anti-CAPRIN-1 antibody used for the purpose ofadministration to human bodies (antibody therapy) is preferably a humanantibody or a humanized antibody for reducing immunogenicity.

An anti-CAPRIN-1 antibody with higher binding affinity for a CAPRIN-1protein on cancer cell surface exerts stronger antitumor activity. Thus,the antibody of the present invention has high binding affinity for theCAPRIN-1 protein and can therefore be expected to have a strongerantitumor effect. Accordingly, the antibody of the present invention isapplicable to a pharmaceutical composition intended for the treatmentand/or prevention of a cancer. Such high binding affinity of theantibody of the present invention is preferably at least 10⁷ M⁻¹, atleast 10⁸ M⁻¹, at least 5×10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 5×10⁹ M⁻¹,at least 10¹⁰ M⁻¹, at least 5×10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least5×10¹¹ M⁻¹, at least 10¹² M⁻¹, or at least 10¹³ M⁻¹, in terms of anassociation constant (affinity constant) Ka (k_(on)/k_(off)), asdescribed above.

The anti-CAPRIN-1 antibody binding to a larger number of CAPRIN-1molecules on cancer cell surface produces stronger antitumor activity.Desirably, the number of CAPRIN-1 molecules in assay using theanti-CAPRIN-1 antibody of the present invention is 10⁴ or more,preferably 10⁵ or more, per cancer cell to which the antibody binds inexpectation of the antitumor effect. Tumor (cancer cells) having a largenumber of CAPRIN-1 molecules on the cell surface is particularlypreferred as a cancer to receive the antibody of the present invention.

<Binding to Antigen-Expressing Cell>

The ability of the antibody to bind to CAPRIN-1 can be determined by useof binding assay using, for example, ELISA, Western blot,immunofluorescence, and flow cytometry analysis, as described inExamples.

<Immunohistochemical Staining>

The antibody that recognizes CAPRIN-1 can be tested for its reactivitywith CAPRIN-1 by an immunohistochemical method well known to thoseskilled in the art using a paraformaldehyde- or acetone-fixed frozensection or paraformaldehyde-fixed paraffin-embedded section of a tissueobtained from a patient during surgical operation or from an animalcarrying a xenograft tissue inoculated with a cell line expressingCAPRIN-1 either spontaneously or after transfection.

For immunohistochemical staining, the antibody reactive with CAPRIN-1can be stained by various methods. For example, the antibody can bevisualized through reaction with a horseradish peroxidase-conjugatedgoat anti-mouse antibody, goat anti-rabbit antibody, or goatanti-chicken antibody.

<Pharmaceutical Composition and Method for Treating and/or PreventingCancer>

A target of the pharmaceutical composition for treatment and/orprevention of a cancer of the present invention is not particularlylimited as long as the target is a cancer (cells) expressing a CAPRIN-1gene.

The terms “tumor” and “cancer” used herein mean malignant neoplasm andare used interchangeably with each other.

The cancer targeted in the present invention is a cancer expressing agene encoding a CAPRIN-1 protein and is preferably breast cancer, kidneycancer, pancreatic cancer, colorectal cancer, lung cancer, brain tumor,gastric cancer, uterine cervix cancer, ovary cancer, prostate cancer,bladder cancer, esophageal cancer, leukemia, lymphoma, fibrosarcoma,mastocytoma, or melanoma.

Specific examples of these cancers include, but are not limited to,breast adenocarcinoma, complex-type breast adenocarcinoma, malignantmixed tumor of mammary gland, intraductal papillary adenocarcinoma, lungadenocarcinoma, squamous cell cancer, small-cell cancer, large-cellcancer, glioma which is tumor of neuroepithelial tissue, ependymoma,neuronal tumor, embryonal neuroectodermal tumor, neurilemmoma,neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma,gastrointestinal lymphoma, alimentary lymphoma, small to mediumcell-type lymphoma, cecal cancer, ascending colon cancer, descendingcolon cancer, transverse colon cancer, sigmoid colon cancer, rectalcancer, epithelial ovarian cancer, germ cell tumor, stromal cell tumor,pancreatic ductal carcinoma, invasive pancreatic ductal carcinoma,pancreatic adenocarcinoma, acinar cell carcinoma, adenosquamouscarcinoma, giant cell tumor, intraductal papillary-mucinous neoplasm,mucinous cystic neoplasm, pancreatoblastoma, serous cystadenocarcinoma,solid-pseudopapillary tumor, gastrinoma, glucagonoma, insulinoma,multiple endocrine neoplasia type-1 (Wermer's syndrome), nonfunctionalislet cell tumor, somatostatinoma, and VIPoma.

The recipient subjects (patients) are preferably mammals, for example,mammals including primates, pet animals, livestock, and sport animalsand are particularly preferably humans, dogs, and cats.

In the case of using the antibody of the present invention as apharmaceutical composition, the pharmaceutical composition can beformulated by a method generally known to those skilled in the art. Forexample, the pharmaceutical composition can be used in the form of aparenteral injection of an aseptic solution or suspension with water orany other pharmaceutically acceptable liquid. For example, thepharmaceutical composition may be formulated with the antibody mixed ina unit dosage form required for generally accepted pharmaceuticalpractice, in appropriate combination with pharmacologically acceptablecarriers or media, specifically, sterilized water, physiological saline,plant oil, an emulsifier, a suspending agent, a surfactant, astabilizer, a flavoring agent, an excipient, a vehicle, a preservative,a binder, etc. The amount of the active ingredient in such a preparationis determined such that an appropriate dose within the prescribed rangecan be achieved.

An aseptic composition for injection can be formulated according toconventional pharmaceutical practice using a vehicle such as injectabledistilled water.

Examples of aqueous solutions for injection include physiologicalsaline, isotonic solutions containing glucose and other adjuvants, forexample, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Thesesolutions may be used in combination with an appropriate solubilizer,for example, an alcohol (specifically, ethanol) or a polyalcohol (e.g.,propylene glycol and polyethylene glycol), or a nonionic surfactant, forexample, polysorbate 80 (TM) or HCO-60.

Examples of oily solutions include sesame oil and soybean oil. Thesesolutions may be used in combination with benzyl benzoate or benzylalcohol as a solubilizer. The solutions may be further mixed with abuffer (e.g., a phosphate buffer solution and a sodium acetate buffersolution), a soothing agent (e.g., procaine hydrochloride), a stabilizer(e.g., benzyl alcohol and phenol), and an antioxidant. The injectionsolutions thus prepared are usually charged into appropriate ampules.

The pharmaceutical composition of the present invention is administeredorally or parenterally, preferably parenterally. Specific examples ofits dosage forms include injections, intranasal administration agents,transpulmonary administration agents, and percutaneous administrationagents. Examples of the injections include intravenous injection,intramuscular injection, intraperitoneal injection, and subcutaneousinjection, through which the pharmaceutical composition can beadministered systemically or locally.

Also, the administration method can be appropriately selected dependingon the age, weight, sex, symptoms, etc. of a patient. The dose of apharmaceutical composition containing the antibody or a polynucleotideencoding the antibody can be selected within a range of, for example,0.0001 to 1000 mg/kg of body weight per dose. Alternatively, the dosecan be selected within a range of, for example, 0.001 to 100000 mg/bodyof a patient, though the dose is not necessarily limited to thesenumeric values. Although the dose and the administration method varydepending on the weight, age, sex, symptoms, etc. of a patient, thoseskilled in the art can appropriately select the dose and the method.

The pharmaceutical composition comprising the antibody of the presentinvention or the fragment thereof can be administered to a subject totreat and/or prevent a cancer, preferably breast cancer, kidney cancer,pancreatic cancer, colorectal cancer, lung cancer, brain tumor, gastriccancer, uterine cervix cancer, ovary cancer, prostate cancer, bladdercancer, esophageal cancer, leukemia, lymphoma, fibrosarcoma,mastocytoma, or melanoma.

The present invention further encompasses a method for treating and/orpreventing cancer, comprising administering the pharmaceuticalcomposition of the present invention in combination with the antitumoragent as exemplified above or a pharmaceutical composition comprisingthe antitumor agent to a subject. The antibody of the present inventionor the fragment thereof may be administered simultaneously with orseparately from the antitumor agent to the subject. In the case ofseparately administering these pharmaceutical compositions, either onemay be administered first or later. Their dosing intervals, doses,administration routes, and the number of doses can be appropriatelyselected by a doctor who is a specialist in cancer therapy. The dosageforms of separate drugs to be administered simultaneously also include,for example, pharmaceutical compositions each formulated by mixing theantibody of the present invention or the fragment thereof or theantitumor agent into a pharmacologically acceptable carrier (or medium).The above descriptions about prescription, formulation, administrationroutes, doses, cancer, etc. as to the pharmaceutical compositions anddosage forms containing the antibody of the present invention are alsoapplicable to any of the above-described pharmaceutical compositions anddosage forms containing the antitumor agent.

Thus, the present invention also provides a combination drug fortreatment and/or prevention of a cancer, comprising the pharmaceuticalcomposition of the present invention and a pharmaceutical compositioncomprising the antitumor agent as exemplified above, and a method fortreating and/or preventing cancer, comprising administering thecombination drug. The present invention also provides a pharmaceuticalcomposition for treatment and/or prevention of cancer, comprising theantibody of the present invention or the fragment thereof and theantitumor agent together with a pharmacologically acceptable carrier.

<Polypeptide and DNA>

The present invention further provides a DNA encoding the antibody ofthe present invention or the fragment (antibody-binding fragment)thereof. Such a DNA may be a DNA encoding the heavy and/or light chainsof the antibody or may be a DNA encoding the heavy and/or light chainvariable regions of the antibody. Such a DNA may also be a DNA encodingeach or a combination of the complementarity determining regions of theantibody. Such a DNA includes, for example, a heavy chain variableregion-encoding DNA comprising nucleotide sequences encoding the aminoacid sequences of SEQ ID NOs: 8, 9, and 10 and a light chain variableregion-encoding DNA comprising nucleotide sequences encoding the aminoacid sequences of SEQ ID NOs: 12, 13, and 14, in the case of theantibody (a).

The complementarity determining regions (CDRs) encoded by the DNA havingthese sequences serve as regions that determine the specificity of theantibody. Sequences encoding the other regions (i.e., constant regionsand framework regions) of the antibody may therefore be sequencesderived from other antibodies. In this context, “other antibodies” alsomay include antibodies derived from non-human organisms but arepreferably those derived from humans from the viewpoint of reducingadverse reactions. Specifically, in the DNA described above, regionsencoding each framework region and each constant region in the heavy andlight chains preferably comprise nucleotide sequences encodingcorresponding human antibody-derived amino acid sequences.

Further examples of the DNA encoding the antibody of the presentinvention include a heavy chain variable region-encoding DNA comprisinga nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11,and a light chain variable region-encoding DNA comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 15, in the caseof the antibody (a). In this context, the nucleotide sequence encodingthe amino acid sequence of SEQ ID NO: 11 is, for example, the nucleotidesequence of SEQ ID NO: 16. The nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 15 is, for example, the nucleotide sequenceof SEQ ID NO: 17. When such a DNA comprises a region encoding eachconstant region in the heavy and light chains, this region preferablycomprises a nucleotide sequence encoding a corresponding humanantibody-derived amino acid sequence (an amino acid sequence of eachconstant region in the heavy and light chains).

These antibody DNAs can be obtained, for example, by the methodsdescribed above or the following method: first, total RNAs are preparedfrom hybridomas producing the antibody of the present invention using acommercially available RNA extraction kit, and cDNAs are synthesizedusing reverse transcriptase and random primers or the like.Subsequently, the antibody-encoding cDNAs are amplified by PCR using, asoligonucleotide primers, conserved sequences of each variable region inknown mouse antibody heavy and light chain genes. Sequences encoding theconstant regions can be obtained by the PCR amplification of knownsequences. The nucleotide sequence of the DNA can be incorporated into aplasmid or a phage for sequencing, for example, and determined accordingto a routine method.

The present invention further provides the following polypeptides andDNAs related to the antibody (a):

(i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 11,and a DNA encoding the polypeptide (e.g., a DNA comprising thenucleotide sequence of SEQ ID NO: 16);

(ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 15,and a DNA encoding the polypeptide (e.g., a DNA comprising thenucleotide sequence of SEQ ID NO: 17);

(iii) a heavy chain CDR polypeptide selected from the group consistingof the amino acid sequences represented by SEQ ID NOs: 8, 9, and 10, anda DNA encoding the polypeptide; and

(iv) a light chain CDR polypeptide selected from the group consisting ofthe amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, anda DNA encoding the polypeptide.

These polypeptides and DNAs can be prepared using gene recombinationtechniques as described above.

SUMMARY OF THE PRESENT INVENTION

The aspects of the present invention described above are summarizedbelow.

(1) An antibody or a fragment thereof which has immunological reactivitywith a partial CAPRIN-1 polypeptide consisting of the amino acidsequence represented by SEQ ID NO: 5 or an amino acid sequence having80% or higher sequence identity to the amino acid sequence.

(2) The antibody or fragment thereof according to (1), wherein theantibody or fragment thereof has cytotoxic activity against a cancercell expressing a CAPRIN-1 protein.

(3) The antibody or fragment thereof according to (1) or (2), whereinthe antibody is a monoclonal antibody or a polyclonal antibody.

(4) The antibody or fragment thereof according to any of (1) to (3),wherein the antibody is a human antibody, a humanized antibody, achimeric antibody, a single-chain antibody, or a multispecific antibody.

(5) The antibody or fragment thereof according to any of (1) to (4),wherein the antibody or fragment thereof comprises a heavy chainvariable region comprising complementarity determining regions of SEQ IDNOs: 8, 9, and 10 (CDR1, CDR2, and CDR3, respectively) and a light chainvariable region comprising complementarity determining regions of SEQ IDNOs: 12, 13, and 14 (CDR1, CDR2, and CDR3, respectively) and hasimmunological reactivity with the CAPRIN-1 protein.

(6) The antibody or fragment thereof according to any of (1) to (5),wherein the antibody or fragment thereof is conjugated with an antitumoragent.

(7) A pharmaceutical composition for treatment and/or prevention of acancer, comprising an antibody or fragment thereof according to any of(1) to (6) as an active ingredient.

(8) The pharmaceutical composition according to (7), wherein the canceris breast cancer, kidney cancer, pancreatic cancer, colorectal cancer,lung cancer, brain tumor, gastric cancer, uterine cervix cancer, ovarycancer, prostate cancer, bladder cancer, esophageal cancer, leukemia,lymphoma, fibrosarcoma, mastocytoma, or melanoma.

(9) A combination drug for treatment and/or prevention of cancer,comprising a pharmaceutical composition according to (7) or (8) and apharmaceutical composition comprising an antitumor agent.

(10) A DNA encoding an antibody or fragment thereof according to any of(1) to (5).

(11) A method for treating and/or preventing cancer, comprisingadministering an antibody or fragment thereof according to any of (1) to(6), a pharmaceutical composition according to (7) or (8), or acombination drug according to (9) to a subject.

EXAMPLES

Hereinafter, the present invention will be described more specificallywith reference to Examples. However, the scope of the present inventionis not intended to be limited by these specific examples.

Example 1 Analysis of CAPRIN-1 Expression in Each Tissue

CAPRIN-1 gene expression in canine and human normal tissues and variouscell lines was examined by RT-PCR according to Example 1(4) ofWO2010/016526. As a result, its strong expression was seen in the testisamong the healthy canine tissues, whereas the expression was seen incanine breast cancer and adenocarcinoma tissues. As a result of alsoconfirming the expression in human tissues, the expression was confirmedonly in the testis among normal tissues, as with the canine CAPRIN-1gene. By contrast, the expression was detected in many types of cancercell lines, including 8 human breast cancer cell lines (ZR75-1, MCF7,T47D, SK-BR-3, MDA-MB-157, BT-20, MDA-MB-231V, and MRK-nu-1) and 4pancreatic cancer cell lines (Capan-2, MIAPaCa-2, Panc-1, and BxPc-3),among cancer cells. These results demonstrated that CAPRIN-1 isexpressed in the breast cancer cell lines and the pancreatic cancer celllines, though its expression is not seen in normal tissues other thanthe testis.

Example 2 Preparation of Mouse Monoclonal Antibody Against CAPRIN-1

(1) Preparation of Mouse Monoclonal Antibody

100 μg of a human CAPRIN-1 protein having the amino acid sequence of SEQID NO: 2 as prepared in Example 3 of WO2010/016526 was mixed with anequal amount of MPL+TDM adjuvant (manufactured by Sigma-Aldrich Corp.).This mixture was used as an antigen solution per mouse. The antigensolution was intraperitoneally administered to each 6-week-old Balb/cmouse (manufactured by Japan SLC, Inc.). Then, 7 boosters were performedevery 1 week to complete immunization. Three days after the final shot,the spleen of each mouse was excised and ground between two sterilizedglass slides. Procedures of washing with PBS(−) (manufactured by NissuiPharmaceutical Co., Ltd.) and removing the supernatant by centrifugationat 1500 rpm for 10 minutes were repeated three times to obtain spleencells. The obtained spleen cells were mixed with mouse myeloma cellsSP2/0 (purchased from ATCC) at a ratio of 10:1. 200 μl of an RPMI1640medium containing 10% FBS was heated to 37° C. and mixed with 800 μl ofPEG1500 (manufactured by Boehringer Ingelheim GmbH), and the PEGsolution thus prepared was added to the cell mixture, which was thenleft standing for 5 minutes for cell fusion. After removal of thesupernatant by centrifugation at 1700 rpm for 5 minutes, the cells weresuspended in 150 ml of an RPMI1640 medium containing 15% FBSsupplemented with 2% equivalent of a HAT solution (manufactured by LifeTechnologies, Inc./Gibco) (HAT selective medium). This suspension wasinoculated to fifteen 96-well plates (manufactured by Thermo FisherScientific Inc./Nunc) at a concentration of 100 μl/well. The spleencells and the myeloma cells were fused by culture at 37° C. for 7 daysunder conditions of 5% CO₂ to obtain hybridomas.

The prepared hybridomas were screened for the binding affinity ofantibodies produced by the hybridomas against CAPRIN-1 proteins as anindicator. A 1 μg/ml solution of the CAPRIN-1 protein prepared by theapproach described in Example 3 of WO2010/016526 was added to a 96-wellplate at a concentration of 100 μl/well and left standing at 4° C. for18 hours. Each well was washed three times with PBS-T. Then, a 0.5%bovine serum albumin (BSA) solution (manufactured by Sigma-AldrichCorp.) was added thereto at a concentration of 400 μl/well and leftstanding at room temperature for 3 hours. The solution in each well wasdiscarded, and each well was washed three times with 400 μl of PBS-T.Then, the culture supernatant of each hybridoma obtained above was addedthereto at a concentration of 100 μl/well and left standing at roomtemperature for 2 hours. Each well was washed three times with PBS-T.Then, HRP-labeled anti-mouse IgG (H+L) antibody (manufactured byInvitrogen Corp.) diluted 5000-fold with PBS was added thereto at aconcentration of 100 μl/well and left standing at room temperature for 1hour. Each well was washed three times with PBS-T. Then, a TMB substratesolution (manufactured by Thermo Fisher Scientific Inc.) was addedthereto at a concentration of 100 μl/well and left standing for 15 to 30minutes to cause color reaction. After the color development, thereaction was terminated by the addition of 1 N sulfuric acid at aconcentration of 100 Owen. The absorbance was measured at 450 nm and 595nm using an absorption spectrometer. As a result, several hybridomasproducing antibodies having high absorbance were selected.

The selected hybridomas were added to a 96-well plate at a density of0.5 cells/well and cultured in the plate. One week later, hybridomasforming single colonies in the wells were observed. The cells in thesewells were further cultured, and the cloned hybridomas were screened forthe binding affinity of antibodies produced by the hybridomas againstCAPRIN-1 proteins as an indicator. A 1 μg/ml solution of the CAPRIN-1proteins prepared in an approach described in Example 3 of WO2010/016526was added to a 96-well plate at a concentration of 100 μl/well and leftstanding at 4° C. for 18 hours. Each well was washed three times withPBS-T. Then, a 0.5% BSA solution was added thereto at a concentration of400 μl/well and left standing at room temperature for 3 hours. Thesolution in each well was discarded, and each well was washed threetimes with 400 μl of PBS-T. Then, the culture supernatant of eachhybridoma obtained above was added thereto at a concentration of 100μl/well and left standing at room temperature for 2 hours. Each well waswashed three times with PBS-T. Then, HRP-labeled anti-mouse IgG (H+L)antibody (manufactured by Invitrogen Corp.) diluted 5000-fold with PBSwas added thereto at a concentration of 100 μl/well and left standing atroom temperature for 1 hour. Each well was washed three times withPBS-T. Then, a TMB substrate solution (manufactured by Thermo FisherScientific Inc.) was added thereto at a concentration of 100 μl/well andleft standing for 15 to 30 minutes to cause color reaction. After thecolor development, the reaction was terminated by the addition of 1 Nsulfuric acid at a concentration of 100 μl/well. The absorbance wasmeasured at 450 nm and 595 nm using an absorption spectrometer. As aresult, 61 hybridoma lines producing monoclonal antibodies reactive withCAPRIN-1 proteins were obtained.

Next, these monoclonal antibodies were screened for antibodies reactivewith the surface of breast cancer cells expressing CAPRIN-1.Specifically, 10⁶ cells of a human breast cancer cell line MDA-MB-231Vwere centrifuged in a 1.5-ml microcentrifuge tube. 100 μl of the culturesupernatant of each hybridoma obtained above was added thereto and leftstanding for 1 hour on ice. After washing with PBS, FITC-labeled goatanti-mouse IgG antibodies (manufactured by Invitrogen Corp.) diluted500-fold with PBS containing 0.1% FBS were added thereto and leftstanding for 1 hour on ice. After washing with PBS, the fluorescenceintensity was measured using FACSCalibur (Becton, Dickinson andCompany). On the other hand, the same operation as above was performedusing the serum of each untreated 6-week-old Balb/c mouse diluted500-fold with a medium for hybridoma culture, instead of the antibodies,to prepare a control. As a result, one monoclonal antibody(anti-CAPRIN-1 antibody #1) having stronger fluorescence intensity thanthat of the control, i.e., reactive with the surface of breast cancercells, was selected.

(2) Identification of CAPRIN-1 Epitope Recognized by Anti-CAPRIN-1Monoclonal Antibody #1

The cancer cell surface-reactive monoclonal antibodies against CAPRIN-1(anti-CAPRIN-1 antibody #1) obtained in the paragraph (1) were used toidentify a CAPRIN-1 epitope region recognized thereby. 93 candidatepeptides each consisting of 12 to 16 amino acids in the amino acidsequence of the human CAPRIN-1 protein were synthesized and eachdissolved at a concentration of 1 mg/ml in DMSO.

Each peptide was dissolved at a concentration of 30 μg/ml in a 0.1 Msodium carbonate buffer solution (pH 9.6). The solution was added at aconcentration of 100 μl/well to a 96-well plate (manufactured by ThermoFisher Scientific Inc./Nunc, product No.: 436006) and left standingovernight at 4° C. The solution in each well was discarded, and 10 mMethanolamine/0.1 M sodium carbonate buffer solution (PH 9.6) was addedthereto at a concentration of 200 μl/well and left standing at roomtemperature for 1 hour. Then, the solution in each well was discarded,and each well was washed twice with PBS containing 0.5% Tween 20 (PBST)to prepare a peptide-immobilized plate.

The cell culture supernatant containing the anti-CAPRIN-1 antibody #1was added at a concentration of 50 μl/well to each plate thus obtained.After shaking at room temperature for 1 hour, the solution in each wellwas discarded, and each well was washed three times with PBST. Next, asecondary antibody solution containing HRP-labeled anti-mouse IgG(manufactured by Invitrogen Corp.) antibodies diluted 3000- to 4000-foldwith PBST was added thereto at a concentration of 50 μl/well. Then, thesolution in each well was discarded, and each well was washed six timeswith PBST.

A TMB substrate solution (manufactured by Thermo Fisher Scientific Inc.)was added thereto at a concentration of 100 μl/well and left standingfor 15 to 30 minutes to cause color reaction. After the colordevelopment, the reaction was terminated by the addition of 1 N sulfuricacid at a concentration of 100 μl/well. The absorbance was measured at450 nm and 595 nm using an absorption spectrometer.

As a result, the polypeptide of SEQ ID NO: 5 was identified as a partialsequence of CAPRIN-1 recognized by the anti-CAPRIN-1 antibody #1obtained in Example 2(1).

(3) Cloning of Variable Region Genes of Anti-CAPRIN-1 Antibody #1

The monoclonal antibodies obtained in Example 2(1) were analyzed fortheir variable region-encoding gene sequences and amino acid sequencesthereof according to the method described in Example 5 of WO2010/016526.As a result, the monoclonal antibody #1 comprised a heavy chain variableregion consisting of the amino acid sequence represented by SEQ ID NO:11 and a light chain variable region consisting of the amino acidsequence represented by SEQ ID NO: 15. A gene sequence encoding theheavy chain variable region of the obtained monoclonal antibody #1 isshown in SEQ ID NO: 16, the amino acid sequence thereof is shown in SEQID NO: 11, a gene sequence encoding the light chain variable regionthereof is shown in SEQ ID NO: 17, and the amino acid sequence thereofis shown in SEQ ID NO: 15.

It was also shown that: the monoclonal antibody #1 obtained in Example2(1) comprises the heavy chain variable region consisting of the aminoacid sequence represented by SEQ ID NO: 11 and the light chain variableregion consisting of the amino acid sequence represented by SEQ ID NO:15, wherein CDR1, CDR2, and CDR3 in the heavy chain variable regionconsist of the amino acid sequences represented by SEQ ID NOs: 8, 9, and10, respectively, and CDR1, CDR2, and CDR3 in the light chain variableregion consist of the amino acid sequences represented by SEQ ID NOs:12, 13, and 14, respectively.

Example 3 Preparation of Polyclonal Antibody Against Partial CAPRIN-1Polypeptide Present on Cancer Cell Surface

In order to obtain polyclonal antibodies against partial CAPRIN-1polypeptides present on cancer cell surface, a polypeptide(CAPRIN-1-derived peptide shown in SEQ ID NO: 5) comprising the epitoperegion for the anti-CAPRIN-1 antibody #1 obtained in Example 2, apolypeptide consisting of a region of amino acid residue numbers 50 to98 in the human CAPRIN-1 amino acid sequence of SEQ ID NO: 2, and apolypeptide consisting of a region of amino acid residue numbers 233 to305 of SEQ ID NO: 2 were synthesized. 1 mg each of these peptides wasmixed as an antigen with an equal volume of an incomplete Freund'sadjuvant (IFA) solution. This mixture was subcutaneously administered toeach rabbit four times every two weeks. Then, blood was collected toobtain antiserum containing each polyclonal antibody. This antiserum wasfurther purified using a protein G carrier (manufactured by GEHealthcare Bio-Sciences Ltd.) and replaced with PBS to obtain polyclonalantibodies against partial CAPRIN-1 polypeptides present on cancer cellsurface. In addition, the serum of a rabbit that received no antigen waspurified using a protein G carrier in the same way as above and used ascontrol antibodies.

Example 4 Analysis of CAPRIN-1 Protein Expression on Cancer CellMembrane Surface

Next, 8 human breast cancer cell lines (ZR75-1, MCF7, T47D, SK-BR-3,MDA-MB-157, BT-20, MDA-MB-231V, and MRK-nu-1) confirmed to have a largelevel of CAPRIN-1 gene expression were examined for their expression ofCAPRIN-1 proteins on the cell surface. 5×10⁵ cells of each human breastcancer cell line thus confirmed to have gene expression were centrifugedin a 1.5-ml microcentrifuge tube. 2 μg (5 μl) each of the polyclonalantibodies against CAPRIN-1-derived peptides (SEQ ID NO: 5) prepared asdescribed above in Example 3 and 95 μl of PBS containing 0.1% fetalbovine serum were added thereto and mixed, and left standing for 1 houron ice. After washing with PBS, the resulting solution was mixed by theaddition of 1 μl of Alexa 488-labeled goat anti-rabbit IgG antibodies(manufactured by Invitrogen Corp.) and 98 μl of PBS containing 0.1%fetal bovine serum (FBS) and left standing for 30 hours on ice. Afterwashing with PBS, the fluorescence intensity was measured usingFACSCalibur (Becton, Dickinson and Company). On the other hand, the sameoperation as above was performed using the control antibodies preparedas described above in Example 3 instead of the polyclonal antibodiesagainst CAPRIN-1-derived peptides to prepare a control. As a result, thecancer cells supplemented with the anti-CAPRIN-1 antibodies allexhibited fluorescence intensity at least 35% stronger than that of thecontrol. This demonstrated that CAPRIN-1 proteins are expressed on thecell membrane surface of the human cancer cell lines. The above rate ofenhancement in fluorescence intensity was indicated by the rate ofincrease in mean fluorescence intensity (MFI) in each cell line andcalculated according to the following expression:

Rate of increase in mean fluorescence intensity(Rate of enhancement influorescence intensity)(%)=((MFI of cells reacted with the anti-CAPRIN-1antibodies)−(Control MFI))/(Control MFI)×100.

Also, the fluorescence intensity was measured in 2 kidney cancer celllines (Caki-1 and Caki-2), a bladder cancer cell line (T24), an ovarycancer cell line (SKOV3), 2 lung cancer cell lines (QG56 and A549), aprostate cancer cell line (PC3), a uterine cervix cancer cell line(SW756), a fibrosarcoma cell line (HT1080), 2 brain tumor cell lines(T98G and U87MG), a gastric cancer cell line (MNK28), 3 colorectalcancer cell lines (Lovo, DLD-1, and HCT-116), and 4 pancreatic cancercell lines (Capan-2, MIAPaCa-2, Panc-1, and BxPC-3) using the sameapproach as above. As a result, all the cancer cells had fluorescenceintensity at least 35% stronger than that of the control.

As with the results obtained above, CAPRIN-1 protein expression oncancer cell membrane surface was also confirmed using the anti-CAPRIN-1antibody #1 obtained in Example 2.

Example 5 Preparation of Human-Mouse Chimeric Monoclonal Antibody

The gene amplification fragment comprising the gene of the heavy chainvariable region of the anti-CAPRIN-1 antibody #1 obtained in Example 2was treated at both ends with restriction enzymes, then purified, andinserted according to a routine method into a pcDNA4/myc-His(manufactured by Invitrogen Corp.) vector already having gene inserts ofa mouse antibody-derived leader sequence and a human IgG₁ H chainconstant region comprising the amino acid sequence of SEQ ID NO: 6.Also, the gene amplification fragment comprising the gene of the lightchain variable region of the anti-CAPRIN-1 antibody #1 was treated atboth ends with restriction enzymes, then purified, and insertedaccording to a routine method into a pcDNA3.1/myc-His (manufactured byInvitrogen Corp.) vector already having gene inserts of a mouseantibody-derived leader sequence and a human IgG₁ L chain constantregion comprising the amino acid sequence of SEQ ID NO: 7.

Next, the recombinant vector having the gene insert of the heavy chainvariable region of the anti-CAPRIN-1 antibody #1 and the recombinantvector having the gene insert of the light chain variable region wereintroduced into CHO-K1 cells (obtained from Riken Cell Bank).Specifically, 2×10⁵ CHO-K1 cells were cultured in 1 ml of a Ham's F12medium (manufactured by Invitrogen Corp.) containing 10% FBS per well ofa 12-well culture plate, and washed with PBS(−). Then, 1 ml of a freshHam's F12 medium containing 10% FBS per well was added thereto. 250 ngeach of the vectors lysed in 30 μl of OptiMEM (manufactured byInvitrogen Corp.) was mixed with 30 μl of Polyfect transfection reagent(manufactured by Qiagen N.V.), and this mixture was added to each well.The CHO-K1 cells cotransfected with the recombinant vectors werecultured in a Ham's F12 medium containing 10% FBS supplemented with 200μg/ml Zeocin (manufactured by Invitrogen Corp.) and 200 μg/ml Geneticin(manufactured by Roche Diagnostics K.K.) and then inoculated to a96-well plate at a density of 0.5 cells/well to prepare cell linesstably producing any of human-mouse chimeric monoclonal antibody #1having the variable regions of the anti-CAPRIN-1 antibody #1, obtainedin Example 2.

Each prepared cell line was cultured for 5 days in a 150-cm² flask at adensity of 5×10⁵ cells/ml using 30 ml of a serum-free OptiCHO medium(manufactured by Invitrogen Corp.) to obtain culture supernatantscontaining the human-mouse chimeric monoclonal antibody #1.

Also, cell lines stably producing human-mouse chimeric comparativeantibodies 1 to 26 were prepared as comparative samples in the same wayas above respectively using the following comparative antibodies:anti-CAPRIN-1 mouse-derived monoclonal antibodies described inWO2010/016526 [a comparative antibody 1 having the heavy chain variableregion of SEQ ID NO: 26 (described therein; the same holds true for thedescription below) and the light chain variable region of SEQ ID NO: 27;a comparative antibody 2 having the heavy chain variable region of SEQID NO: 28 and the light chain variable region of SEQ ID NO: 29; acomparative antibody 3 having the heavy chain variable region of SEQ IDNO: 30 and the light chain variable region of SEQ ID NO: 31; acomparative antibody 4 having the heavy chain variable region of SEQ IDNO: 32 and the light chain variable region of SEQ ID NO: 33; acomparative antibody 5 having the heavy chain variable region of SEQ IDNO: 34 and the light chain variable region of SEQ ID NO: 35; acomparative antibody 6 having the heavy chain variable region of SEQ IDNO: 36 and the light chain variable region of SEQ ID NO: 37; acomparative antibody 7 having the heavy chain variable region of SEQ IDNO: 38 and the light chain variable region of SEQ ID NO: 39; acomparative antibody 8 having the heavy chain variable region of SEQ IDNO: 40 and the light chain variable region of SEQ ID NO: 41; acomparative antibody 9 having the heavy chain variable region of SEQ IDNO: 42 and the light chain variable region of SEQ ID NO: 43; acomparative antibody 10 having the heavy chain variable region of SEQ IDNO: 44 and the light chain variable region of SEQ ID NO: 45; and acomparative antibody 11 having the heavy chain variable region of SEQ IDNO: 46 and the light chain variable region of SEQ ID NO: 47],anti-CAPRIN-1 monoclonal antibodies described in WO2011/096517 [acomparative antibody 12 having the heavy chain variable region of SEQ IDNO: 43 (described therein; the same holds true for the descriptionbelow) and the light chain variable region of SEQ ID NO: 47; and acomparative antibody 13 having the heavy chain variable region of SEQ IDNO: 43 and the light chain variable region of SEQ ID NO:], anti-CAPRIN-1monoclonal antibodies described in WO2011/096528 [a comparative antibody14 having the heavy chain variable region of SEQ ID NO: 43 (describedtherein; the same holds true for the description below) and the lightchain variable region of SEQ ID NO: 47; a comparative antibody 15 havingthe heavy chain variable region of SEQ ID NO: 51 and the light chainvariable region of SEQ ID NO: 55; a comparative antibody 16 having theheavy chain variable region of SEQ ID NO: 59 and the light chainvariable region of SEQ ID NO: 63; a comparative antibody 17 having theheavy chain variable region of SEQ ID NO: 76 and the light chainvariable region of SEQ ID NO: 80; a comparative antibody 18 having theheavy chain variable region of SEQ ID NO: 84 and the light chainvariable region of SEQ ID NO: 88; and a comparative antibody 19 havingthe heavy chain variable region of SEQ ID NO: 92 and the light chainvariable region of SEQ ID NO: 96], an anti-CAPRIN-1 monoclonal antibodydescribed in WO2011/096519 [a comparative antibody 20 having the heavychain variable region of SEQ ID NO: 42 (described therein; the sameholds true for the description below) and the light chain variableregion of SEQ ID NO: 46], anti-CAPRIN-1 monoclonal antibodies describedin WO2011/096533 [a comparative antibody 21 having the heavy chainvariable region of SEQ ID NO: 43 (the same holds true for thedescription below) and the light chain variable region of SEQ ID NO: 51;a comparative antibody 22 having the heavy chain variable region of SEQID NO: 47 and the light chain variable region of SEQ ID NO: 51; and acomparative antibody 23 having the heavy chain variable region of SEQ IDNO: 63 and the light chain variable region of SEQ ID NO: 67], andanti-CAPRIN-1 monoclonal antibodies described in WO2011/096534 [acomparative antibody 24 having the heavy chain variable region of SEQ IDNO: 43 (described therein; the same holds true for the descriptionbelow) and the light chain variable region of SEQ ID NO: 47; acomparative antibody 25 having the heavy chain variable region of SEQ IDNO: 43 and the light chain variable region of SEQ ID NO: 51; and acomparative antibody 26 having the heavy chain variable region of SEQ IDNO: 63 and the light chain variable region of SEQ ID NO: 67]. Eachprepared cell line was cultured for 5 days in a 150-cm² flask at adensity of 5×10⁵ cells/ml using 30 ml of a serum-free OptiCHO medium(manufactured by Invitrogen Corp.) to obtain culture supernatantscontaining any of the human-mouse chimeric comparative monoclonalantibodies 1 to 26.

Example 6 Expression of CAPRIN-1 on Surface of Various Cancer CellsUsing Anti-CAPRIN-1 Monoclonal Antibody

Next, the 8 human breast cancer cell lines (ZR75-1, MCF7, T47D, SK-BR-3,MDA-MB-157, BT-20, MDA-MB-231V, and MRK-nu-1), the 2 kidney cancer celllines (Caki-1 and Caki-2), the bladder cancer cell line (T24), the ovarycancer cell line (SKOV3), the 2 lung cancer cell lines (QG56 and A549),the prostate cancer cell line (PC3), the uterine cervix cancer cell line(SW756), the fibrosarcoma cell line (HT1080), the 2 brain tumor celllines (T98G and U87MG), the gastric cancer cell line (MNK28), the 3colorectal cancer cell lines (Lovo, DLD-1, and HCT-116), and the 4pancreatic cancer cell lines (Capan-2, MIAPaCa-2, Panc-1, and BxPC-3)confirmed to have CAPRIN-1 gene expression were examined for theirexpression of CAPRIN-1 proteins on the cell surface using the culturesupernatants containing the anti-CAPRIN-1 antibody #1 obtained inExample 2. 10⁶ cells of each cell line were centrifuged in each 1.5-mlmicrocentrifuge tube. Each culture supernatant (100 μl) containing theantibody was added to the tube and left standing for 1 hour on ice.After washing with PBS, FITC-labeled goat anti-mouse IgG (H+L) antibody(manufactured by Jackson ImmunoResearch Laboratories, Inc.) diluted withPBS containing 0.1% FBS was added thereto and left standing at 4° C. for30 minutes. After washing with PBS, the fluorescence intensity wasmeasured using FACSCalibur (Becton, Dickinson and Company). The negativecontrol used was cells reacted only with secondary antibodies. As aresult, the anti-CAPRIN-1 antibody #1 exhibited reactivity withfluorescence intensity at least 30% stronger than that of the negativecontrol. This demonstrated that CAPRIN-1 proteins are expressed on thecell membrane surface of the human cancer cell lines. The above rate ofenhancement in fluorescence intensity was indicated by the rate ofincrease in mean fluorescence intensity (MFI) in each cell line andcalculated according to the following expression:

Rate of increase in mean fluorescence intensity(Rate of enhancement influorescence intensity)(%)=((MFI of cells reacted with the anti-CAPRIN-1antibodies)−(Control MFI))/(Control MFI)×100.

Example 7 Antitumor Activity Against Cancer Cell of Antibody AgainstCAPRIN-1-Derived Peptide (SEQ ID NO: 5)

In order to evaluate each antibody against the CAPRIN-1-derived peptide(SEQ ID NO: 5) for the strength of its cytotoxicity against cancer cellsexpressing CAPRIN-1, ADCC activity was determined. The polyclonalantibodies against the peptide (SEQ ID NO: 5) prepared in Example 3 wereused in this evaluation. Similar evaluation was conducted usingpolyclonal antibodies against other human CAPRIN-1-derived peptides(polyclonal antibodies against amino acid residue numbers 50 to 98 inthe amino acid sequence of SEQ ID NO: 2 of human CAPRIN-1 and polyclonalantibodies against amino acid residue numbers 233 to 305, which wereprepared in Example 3) as antibodies to be compared and the rabbitserum-derived control antibodies prepared in Example 3 as a negativecontrol.

10⁶ cells each of the human breast cancer cell line MDA-MB-231V, thehuman colorectal cancer cell line DLD-1, the human pancreatic cancercell line Capan-2, and the human lung cancer cell line QG56 confirmed tohave CAPRIN-1 expression were collected into a 50-ml centrifuge tube, towhich 100 μCi of chromium 51 was then added, followed by incubation at37° C. for 2 hours. Then, the cells were washed three times with anRPMI1640 medium containing 10% fetal calf serum and added at a densityof 2×10³ cells/well to each 96-well V-bottom plate. The polyclonalantibodies against the human CAPRIN-1-derived peptide (SEQ ID NO: 5) andtwo types of polyclonal antibodies against other human CAPRIN-1-derivedpeptides (polyclonal antibodies against amino acid residue numbers 50 to98 in SEQ ID NO: 2 of human CAPRIN-1 and polyclonal antibodies againstamino acid residue numbers 233 to 305) as described above wereseparately added thereto at a concentration of 1 μg/well. Lymphocytesseparated from human peripheral blood according to a routine method werefurther added thereto at a density of 4×10⁵ cells/well and cultured at37° C. for 4 hours under conditions of 5% CO₂. After the culture, theamount of chromium (Cr) 51 released from damaged cancer cells wasmeasured in the culture supernatant to calculate the ADCC activityagainst the cancer cells of the polyclonal antibodies against each humanCAPRIN-1-derived peptide. As a result, all the polyclonal antibodiesobtained by immunization with the partial peptides of human CAPRIN-1having an amino acid sequence of amino acid residue numbers 50 to 98 oramino acid residue numbers 233 to 305 of SEQ ID NO: 2 of human CAPRIN-1had activity less than 8% against the human breast cancer cell lineMDA-MB-231V, the human colorectal cancer cell line DLD-1, the humanpancreatic cancer cell line Capan-2, and the human lung cancer cell lineQG56. By contrast, the groups supplemented with the polyclonalantibodies against the human CAPRIN-1-derived peptide (SEQ ID NO: 5)were confirmed to have 27% or higher cytotoxic activity against all thecancer cell lines. The negative control antibodies had activity lessthan 5% against all the cancer cells. These results demonstrated thatthe antibody against CAPRIN-1 shown in SEQ ID NO: 5 exerts strongcytotoxic activity against cancer cells expressing CAPRIN-1.

These results about cytotoxic activity were obtained by: mixing theantibody against CAPRIN-1 used in the present invention, lymphocytes,and 2×10³ cells of each cancer cell line with incorporated chromium 51,as described above: culturing the cells for 4 hours; after the culture,measuring the amount of chromium 51 released into the medium; andcalculating the cytotoxic activity against each cancer cell lineaccording to the following expression*:

*Expression: Cytotoxic activity(%)=Amount of chromium 51 released fromthe target cells supplemented with the antibody against CAPRIN-1 andlymphocytes/Amount of chromium 51 released from target cellssupplemented with 1 N hydrochloric acid×100.

The human-mouse chimeric monoclonal antibody obtained in Example 5 wereevaluated for their cytotoxic activity against human cancer cells. Theculture supernatant of each cell line producing any of the antibodieswas purified using Hitrap Protein A Sepharose FF (manufactured by GEHealthcare Bio-Sciences Ltd.). After replacement with PBS(−), thesolution was filtered through a 0.22-μm filter (manufactured byMillipore Corp.). The resulting antibody was used for activity assay.10⁶ cells each of the human breast cancer cell line MDA-MB-231V, thehuman colorectal cancer cell line DLD-1, the human pancreatic cancercell line Capan-2, and the human lung cancer cell line QG56 werecollected into a 50-ml centrifuge tube, to which 100 μCi of chromium 51was then added, followed by incubation at 37° C. for 2 hours. Then, thecells were washed three times with an RPMI1640 medium containing 10% FBSand added at a density of 2×10³ cells/well to each 96-well V-bottomplate to prepare target cells. The purified antibodies (human-mousechimeric anti-CAPRIN-1 antibody #1) and the human-mouse chimericcomparative monoclonal antibodies 1 to 26 obtained in Example 5 wereeach added thereto at a concentration of 0.75 μg/well. A cell populationcontaining human NK cells was separated using a routine method fromhuman peripheral blood lymphocytes prepared according to a routinemethod. The cell population containing human NK cells that was used inthis evaluation was prepared as follows: human peripheral bloodmononuclear cells separated using a specific gravity separation solutionHistopaque for peripheral blood mononuclear cell separation(Sigma-Aldrich Corp.) were reacted with FITC fluorescent dye-labeledantibodies (anti-human CD3 antibody, anti-human CD20 antibody,anti-human CD19 antibody, anti-human CD11c antibody, or anti-HLA-DRantibody (Becton, and Dickinson and Company)), and a cell populationcontaining NK cells unstained with the antibodies was separated using acell sorter (FACS Vantage SE (Becton, and Dickinson and Company)) orhuman NK cell separation kit (manufactured by Miltenyi Biotec K.K.). Theseparated cell population containing NK cells was added to the plate ata density of 2×10⁵ cells/well and cultured at 37° C. for 4 hours underconditions of 5% CO₂. After the culture, the amount of chromium 51released from damaged tumor cells was measured in the culturesupernatant to calculate the cytotoxic activity of each anti-CAPRIN-1antibody against the cancer cells. The negative control used was cellssupplemented with isotype control antibodies. As a result, the isotypecontrol antibodies used had cytotoxic activity of less than 5% againstall of the cancer cell lines, and the human-mouse chimeric comparativemonoclonal antibodies 1 to 26 used had cytotoxic activity of less than5% against MDA-MB-231V, less than 8% against DLD-1, less than 10%against Capan-2, and less than 10% against QG56. By contrast, thehuman-mouse chimeric anti-CAPRIN-1 antibody #1 had cytotoxic activity of12% or higher against MDA-MB-231V, 22% or higher against DLD-1, 28% orhigher against Capan-2, and 21% or higher against QG56. Likewise, theisotype control antibodies used and the comparative antibodies 1 to 26used had cytotoxic activity less than 4% against all of other cancercells, breast cancer cell lines T47D, Hs578T, BT-20, SK-BR-3, MCF7, andMRK-nu-1, a glioma cell line T98G, a lung cancer cell line A549, akidney cancer cell line Caki-1, a uterine cervix cancer cell line SW756,a bladder cancer cell line T24, a gastric cancer cell line MKN28, acolorectal cancer cell line SW480, a leukemia cell line AML5, and alymphoma cell line Ramos. By contrast, the human-mouse chimericmonoclonal antibody was confirmed to have 10% or higher cytotoxicactivity against these cell lines. These results showed that theantibodies against the CAPRIN-1-derived peptide shown in SEQ ID NO: 5damage CAPRIN-1-expressing cancer cells through their ADCC activity, anddemonstrated that the human-mouse chimeric anti-CAPRIN-1 antibody #1exhibits stronger cytotoxic activity against human cancer cells thanthat of the comparative antibodies 1 to 26.

These results about cytotoxic activity were obtained by: mixing theantibody against CAPRIN-1 used in the present invention, lymphocytes(cell population containing NK cells), and 2×10³ cells of each cancercell line with incorporated chromium 51, as described above: culturingthe cells for 4 hours; after the culture, measuring the amount ofchromium 51 released into the medium; and calculating the cytotoxicactivity against each cancer cell line according to the followingexpression*:

*Expression: Cytotoxic activity(%)=Amount of chromium 51 released fromthe target cells supplemented with the antibody against CAPRIN-1 andlymphocytes(cell population containing NK cells)/Amount of chromium 51released from target cells supplemented with 1 N hydrochloric acid×100.

Example 8 The Number of CAPRIN-1 Molecules on Surface of Various CancerCells Recognized by Anti-CAPRIN-1 Antibody #1

A human breast cancer cell line (MDA-MB-231V), a kidney cancer cell line(Caki-1), a bladder cancer cell line (T24), an ovary cancer cell line(SKOV3), lung cancer cell lines (QG56 and A549), a pancreatic cancercell line (Capan-2), a prostate cancer cell line (PC3), a uterine cervixcancer cell line (SW756), a fibrosarcoma cell line (HT1080), a braintumor cell line (T98G), a gastric cancer cell line (MKN28), colorectalcancer cell lines (Lovo and DLD-1), a leukemia cell line (AML5), and alymphoma cell line (Ramos) were examined using an assay kit “QIFIKIT”for the number of molecules (manufactured by Dako Japan Inc.) for thenumber of CAPRIN-1 molecules on their cell surface recognized by theanti-CAPRIN-1 antibody #1. Similarly, the number of CAPRIN-1 moleculeson the surface of these various cancer cells was also examined using theanti-CAPRIN-1 comparative monoclonal antibodies 1 to 26 prepared inExample 5.

According to the protocol attached to the kit, each antibody(anti-CAPRIN-1 antibodies #1 and comparative antibodies 1 to 26) wasdiluted into 5 μg/ml (in terms of final concentration) with PBS, andthis dilution was added to each cell line and reacted for 30 minutes.After washing with PBS, fluorescently labeled anti-mouse IgG antibodiesattached to the kit were added as secondary antibodies, together withcalibration beads attached to the kit, to each cell line and leftstanding for 45 minutes on ice. Each cell line and the calibration beadswere washed with PBS. Then, the fluorescence intensity was measuredusing FACSCalibur (Becton, Dickinson and Company) to obtain a meanfluorescence intensity value (mean). Also, a mean fluorescence intensityvalue (mean) was obtained by the same assay as above for the comparativeantibodies. The negative control used was cells reacted with isotypecontrol antibodies, and a mean was also obtained. Each mean fluorescenceintensity value (mean) was used to calculate the number of moleculesaccording to the protocol attached to the kit. As a result, the numberof CAPRIN-1 molecules on the surface of various cancer cells recognizedby the anti-CAPRIN-1 monoclonal antibody and the comparative antibodies12 to 26 was 10⁵ or more per cell for all the examined human cancer celllines. On the other hand, the number of molecules recognized by thecomparative antibodies 1 to 11 was less than 10⁵ per cell.

INDUSTRIAL APPLICABILITY

The antibody of the present invention is useful for the treatment and/orprevention of cancer.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. An antibody or a fragment thereof which has immunological reactivitywith a partial CAPRIN-1 polypeptide having the amino acid sequencerepresented by SEQ ID NO: 5 or an amino acid sequence having 80% orhigher sequence identity to the amino acid sequence.
 2. The antibody orfragment thereof according to claim 1, wherein the antibody or fragmentthereof has cytotoxic activity against a cancer cell expressing aCAPRIN-1 protein.
 3. The antibody or fragment thereof according to claim1, wherein the antibody is a monoclonal antibody or a polyclonalantibody.
 4. The antibody or fragment thereof according to claim 1,wherein the antibody is a human antibody, a humanized antibody, achimeric antibody, a single-chain antibody, or a multispecific antibody.5. The antibody or fragment thereof according to claim 1, wherein theantibody or fragment thereof comprises a heavy chain variable regioncomprising complementarity determining regions of SEQ ID NOs: 8, 9, and10 (CDR1, CDR2, and CDR3, respectively) and a light chain variableregion comprising complementarity determining regions of SEQ ID NOs: 12,13, and 14 (CDR1, CDR2, and CDR3, respectively) and has immunologicalreactivity with the CAPRIN-1 protein.
 6. The antibody or fragmentthereof according to claim 1, wherein the antibody or fragment thereofis conjugated with an antitumor agent.
 7. A pharmaceutical compositionfor treatment and/or prevention of a cancer, comprising an antibody orfragment thereof according to claim 1 as an active ingredient.
 8. Thepharmaceutical composition according to claim 7, wherein the cancer isbreast cancer, kidney cancer, pancreatic cancer, colorectal cancer, lungcancer, brain tumor, gastric cancer, uterine cervix cancer, ovarycancer, prostate cancer, bladder cancer, esophageal cancer, leukemia,lymphoma, fibrosarcoma, mastocytoma, or melanoma.
 9. A combination drugfor treatment and/or prevention of cancer, comprising a pharmaceuticalcomposition according to claim 7 and a pharmaceutical compositioncomprising an antitumor agent.
 10. A DNA encoding an antibody orfragment thereof according to claim
 1. 11. A method for treating and/orpreventing cancer, comprising administering an antibody or fragmentthereof according to claim 1 to a subject.
 12. The antibody or fragmentthereof according to claim 2, wherein the antibody is a monoclonalantibody or a polyclonal antibody.
 13. The antibody or fragment thereofaccording to claim 2, wherein the antibody is a human antibody, ahumanized antibody, a chimeric antibody, a single-chain antibody, or amultispecific antibody.
 14. The antibody or fragment thereof accordingto claim 3, wherein the antibody is a human antibody, a humanizedantibody, a chimeric antibody, a single-chain antibody, or amultispecific antibody.
 15. The antibody or fragment thereof accordingto claim 2, wherein the antibody or fragment thereof comprises a heavychain variable region comprising complementarity determining regions ofSEQ ID NOs: 8, 9, and 10 (CDR1, CDR2, and CDR3, respectively) and alight chain variable region comprising complementarity determiningregions of SEQ ID NOs: 12, 13, and 14 (CDR1, CDR2, and CDR3,respectively) and has immunological reactivity with the CAPRIN-1protein.
 16. The antibody or fragment thereof according to claim 3,wherein the antibody or fragment thereof comprises a heavy chainvariable region comprising complementarity determining regions of SEQ IDNOs: 8, 9, and 10 (CDR1, CDR2, and CDR3, respectively) and a light chainvariable region comprising complementarity determining regions of SEQ IDNOs: 12, 13, and 14 (CDR1, CDR2, and CDR3, respectively) and hasimmunological reactivity with the CAPRIN-1 protein.
 17. The antibody orfragment thereof according to claim 4, wherein the antibody or fragmentthereof comprises a heavy chain variable region comprisingcomplementarity determining regions of SEQ ID NOs: 8, 9, and 10 (CDR1,CDR2, and CDR3, respectively) and a light chain variable regioncomprising complementarity determining regions of SEQ ID NOs: 12, 13,and 14 (CDR1, CDR2, and CDR3, respectively) and has immunologicalreactivity with the CAPRIN-1 protein.
 18. The antibody or fragmentthereof according to claim 2, wherein the antibody or fragment thereofis conjugated with an antitumor agent.
 19. The antibody or fragmentthereof according to claim 3, wherein the antibody or fragment thereofis conjugated with an antitumor agent.
 20. The antibody or fragmentthereof according to claim 4, wherein the antibody or fragment thereofis conjugated with an antitumor agent.